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Mechanistic studies of peptidylglycine alpha-amidating monooxygenase (PAM)

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Title:
Mechanistic studies of peptidylglycine alpha-amidating monooxygenase (PAM)
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English
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McIntyre, Neil R
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Subjects / Keywords:
Hydroxylation
Amidation
Imino-oxy
Acylglycine
Dissertations, Academic -- Chemistry -- Doctoral -- USF   ( lcsh )
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non-fiction   ( marcgt )

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Summary:
ABSTRACT: Peptide hormones are responsible for cellular functions critical to the survival of an organism. Approximately 50% of all known peptide hormones are post-translationally modified at their C-terminus. Peptidylglycine alpha-amidating monooxygenase (PAM) is a bi-functional enzyme which catalyzes the activation of peptide pro-hormones. PAM also functionalizes long chain N-acylglycines suggesting a potential role in signaling as their respective fatty acid amides. As chain length increases for N-acylglycines so does the catalytic efficiency. This effect was probed further by primary kinetic isotope effects and molecular dynamics to better resolve the mechanism for improved catalytic function. The °KIE showed a linear decrease with increasing chain length. Neither the minimal kinetic mechanism nor the maximal rate for substrate oxidation was observed to be altered by substrate hydrophobicity.It was concluded that KIE suppression was a function of 'Pre-organization' - more efficient degenerate wave function overlap between C-H donor and Cu(II)-superoxo acceptor with increased chain length. Substrate activation is believed to be facilitated by a Cu(II)-superoxo complex formed at CusubscriptM. Benzaldehyde imino-oxy acetic acid undergoes non-enzymatic O-dealkylation to the corresponding oxime and glyoxylate products. This phenomena was further studied using QM/MM methodology using different Cu/O species to determine which best facilitated the dealkylation event. It was determined that radical recombination between a Cu(II)-oxyl and a substrate radical to form an unstable copper-alkoxide intermediate was best suited to carry out this reaction. Structure-function analysis was used to rationalize the electronic features which made a variety of diverse imino-oxy acetic acid analogues such unexpectedly good PAM substrates (10⁴⁻⁵ M⁻¹s⁻¹).To observe the effect oxygen insertion and placement had on substrates between N-benzoylglycine and benzaldehyde imino-oxy acetic acid structures, PAM activity was correlated with NBO/MEP calculations on selected PHM-docked structures. This work concluded that the imino-oxy acetic acid was a favored substrate for PAM because its oxime electronically is very similar to the amide present in glycine-extended analogues.
Thesis:
Dissertation (Ph.D.)--University of South Florida, 2008.
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Includes bibliographical references.
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by Neil R. McIntyre.
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Document formatted into pages; contains 247 pages.
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Mechanistic Studies of Peptidylglyc ine Alpha-Amidating Monooxygenase (PAM) by Neil R. McIntyre A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemistry College of Arts and Sciences University of South Florida Major Professor: David Merkler, Ph.D. Edward Turos, Ph.D. Peter Zhang, Ph.D. Nicole Horenstein, Ph.D. Date of Approval: March 26, 2008 Keywords: hydroxylation, amida tion, imino-oxy, acylglycine Copyright 2008 Neil R. McIntyre

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Acknowledgments Growing up, I always loved sports and the personalities within. One of my favorite quotes was by Bill Russell, a hall-of-fame center for the Boston Celtics who collected more championship rings than fingers (11 in total). His motivation to succeed was simple I always wanted to be remember ed, not recalled This phrase played in my mind many times over my years in the Merkler lab. Professor Merkler is a big idea thinker and may be the most original scientis t I have known our area of science. As a supervisor, this allowed him to meet you at any level of any idea to make you understand something fundamental about your theory. Ob viously, I have the utmost respect for his intellect and sought to absorb as many lab t echniques as possible, simply so we could have these dynamic talks about science. Beyond Dr. Merkler, othe r researchers have been very influential to me: Drs. Potte r, Grossman, Owen, Turos, and Space. Writing a dissertation was a challenge I would never want to endure again. Interpretation of the phrase you only have one dissertation inside you is now very clear to me. I also believe that your family and friends can only endure a single dissertation. My family has been incredibly supportive of me over the last half-decade. My mother and father have always beli eved in me, helping me emotionally and financially to complete the bulk of this work. As a result this work is ultimately dedicated to them. Tha nks to my sister, Shannon, for never asking me when I would be done writing and my lab-family EDL and SEC for many enga ging conversations about science (along with many laughs ) at Savy Jacks each Saturday. The Tampa judo club has also provided me an environment to redu ce some frustration over the years. Finally, I need to thank MRF for beli eving in me over the last y ear and giving me something I could never learn in a chemistry lab.

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i Table of Contents List of Tables v List of Figures vi List of Schemes ix List of Spectra xii List of Abbreviations xiv Abstract xv Chapter One: Introduction 1 The -Amidating Enzyme 1 Structural Features of PHM and D M 2 Peptidylglycine Amidoglycolate Lyase (PAL) 4 Review of Oxygen Activation Among Monooxygenases 6 Non-coupled monooxygenases 15 Sequence Homology Between PHM and D M 16 Substrate Speci ficity Among Glycine-Extended Peptides 19 Electron Transfer between CuH and CuM 20 PHM Structure 20 Copper Domain Geometry 23 Background to Postulated Mechanisms 26 Minimal Kinetic Mechanism 28 Role of Tunneling in C-H Bond Cleavage 28 Computational Studies 28 Evidence of CuII-superoxo Intermediate 30 Evidence for a Radical Substrate Intermediate 30 Postulated Reaction Mechanism for PHM/D M 31 Introduction to Dissertation Chapters 37 References 39 Chapter Two: Substrate Pr e-Organization in PHM 48 Introduction 48 Materials and Methods 57 Materials 57 Methods 57

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ii Synthesis of N -Acylglycines, including the -Dideutero-Analogues 57 Steady State Kinetics R eaction Conditions 58 Determination the O2-Dependence of the Kinetic Isotope Effects 58 Viscosity Dependence of the PAM-Catalyzed Oxidation of N -Acylglycines 59 Predicted Docking Conformations of N -Acylglycines within the PHM Active-Site 62 Molecular Dynamics using NAMD and Alchemical Free Energy Perturbation 62 i) Protein Minimization and Equilibration 62 ii) Alchemi cal Free Energy Perturbation Method 64 Equilibrium Dynamics Studies of PHM-Substrate Complexes 66 Results 67 Characterization of th e Synthesized N-Acylglycines and [-2H2]N -Acylglycines 67 Steady-State Kinetic Data 71 Minimal Kinetic Mechanism 73 Viscosity Effects 79 Chain Length Effect Under Saturating Oxygen Conditions 81 Computational Chemistry 82 i) Predicted Docking Conformations 82 ii) Equilibration, Alchemical Free Energy Perturbation (AFEP) and Equilibrium Dynamics 82 Discussion 87 Minimal Kinetic Mechanism from Kinetic Isotope Effects 87 Interpretation of the VMAX Isotope Kinetic Isotope Effect 89 Structure Dependenc e on Binding Mode 90 Evidence for conformational flexibility 94 Source of the Is otope Effect in Tunneling Reactions 95 Application of Pre-Organization 96 Conclusion 96 Future work 97 Analogous Pre-Organization in D M? 98 References 99 Chapter Three: Mechanism fo r Substrate hydroxylation 105 Introduction 105 Structures 105 Reaction Mechanism and Model Studies 108 CopperII Superoxo and High-Valent Copper-Oxo

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iii Species in PHM/D M 109 Salient Mechanis tic Characteristics 113 Probing Substrate Oxidation Through De-Alkylation 115 Materials 117 Recombinant PAM 117 Methods 117 Synthesis of Benzaldehyde imino-oxy acetic acid 117 Steady State Kinetic Oximetry 118 Glyoxylate Stoichiometry 119 Product Characterization 120 Oxygen Stoichiometry 120 Stability of Putative -Hydroxy-Benzaldehyde Imino-Oxy Acetic Acid 121 Non-competitive Isotope Effects 122 Quantum Mechanical/Molecular Mechanics Reaction Coordinate 125 Results 129 Reaction Stoichiometry, Product Identification and PHM Product Stability 129 Stability of the Benzaldehyde Imino-Oxy Acetic Acid Oxidation Product 132 Kinetic Isotope Effects 135 QM/MM Reaction Coordinate 139 Substrate Dealkylation 146 Discussion 146 De-alkylation is Non-Enzymatic 146 Kinetic Mechanism 147 Substrate Oxidation and Product Release are Uncoupled 152 Conclusion 154 Future Aspects to This Study 156 References 157 Chapter Four: Structure-Function of the Im ino-Oxy Acetic Acid 164 Introduction 164 Therapeutic Targeting of PAM 166 Theoretical Tools for Structure-Function Analysis 168 Molecular Electro static Potential versus Natural Bond Order Population Analysis 168 Hyperconjugation 170 Natural Population Analysis versus Mulliken 171 Second Order Perturbation Theory 171 Bents Rule 172 Precedence for NBO Analysis 172 Structure-Function of the Imino-Oxy Acetic Acid 174

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iv Material and Methods 176 Materials 176 Methods 176 Synthesis of aryl imino-oxy acetic acids 176 -Dideutero Acetophenoneand -Naphthaldehyde imino-oxy acetic acid 176 para-(5-Dimethylaminonapthalene-1sulfonamido) acetophenone imino-oxy acetic acid 177 Steady State kinetic Oxygraph 178 Substrate Analysis 178 Acetylglycine Oxidation 179 Inhibition of N -dansyl-tyr-val-gly amidation by imino-oxy acetic acid derivatives 180 Computation 181 Electr onic structure calculations 182 Supplementary Note 182 Results 182 Steady State Kine tics of Imino-Oxy Acetic Acid Derivatives 182 Structure-Function Analysis 187 Electronic Structure Calculations 187 Glycine versus Carboxymethylhydroxylamine 187 N -Benzoylglycine versus O -acetylbenzoHydroxamic Acid 188 Phenylacet ylamino-acetic Acid versus Phenylacetylamino-oxy acetic Acid 189 (Benzylideneamino)-Acetic Acid versus Benzaldehyde Imino-Oxy Acetic Acid 190 Discussion 194 Imino-Oxy Acetic Acids 194 Effect of Oxygen-Insertion 194 Conclusion 198 Potential Applications of NBO Analysis 200 Experimental 201 References 207 Chapter Five: NMR Spectra of Imino-Oxy Acetic acids and N -acylglycines 208 Appendices 168 Appendix A: Abbreviations 169 About the Author End Page

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v List of Tables Table 1.1 Sequence homology comparing ac tive site residues from PHM to analogous residues in D M 17 Table 2.1 The Non-competitive deuterium kinetic isotope effect as a function of N -acylglycine chain length 72 Table 2.2 Kinetic parameters for N -acetylglycine and [ -2H2]-N-acetylglycine fit to the equilibrium-preferred rate expression 76 Table 2.3 Apparent kinetic parameters for N -acetylglycine and [-2H2]N acetylglycine at varying oxygen concentrations 77 Table 2.4 Kinetic parameters for (VMAX/KM)OXYGEN determined as a function of N -acylglycine chain length. 81 Table 3.1 Stoichiometry of the enzy matic [oxygen] versus [Benzaldehyde imino-oxy acetic acid] consumption measured below the ambient concentration of dissolved oxygen. 131 Table 3.2 Kinetic parameters fit to a steady-state preferred rate Expression fo r benzaldehyde imino-oxy acetic acid variation with oxygen concentration held c onstant. 135 Table 3.3 Kinetic parameters fit to a steady-state preferred rate Expression fo r oxygen variation with benzaldehyde imino-oxy acetic acid concentration held cons tant. 135 Table 3.4 Kinetic parameters dete rmined from the steady-state preferred bi-substrate Michaelis-Menton eq uation 138 Table 4.1 Kinetic parameters for PAM-dependent oxidation of substrates. 187 Table 4.2 Inhibition constant (KI) determination for imino-oxy acetic acid and hydroxamic acid compounds. 189 Table 4.3 Apparent -di-deutero kinetic isotope effect DKIE,APP for acetophenone imino-oxy acetic acid and -naphthaldehyde imino-oxy acetic acid measured under ambient oxygen tension 189 Table 4.4 Second-order perturbation theo ry values for structure-function molecules determined from natural bond orbital analysis (NBO). 197

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vi List of Figures Figure 1.1 Structure comparison of flavin and pterin. 8 Figure 1.2 Consensus sequence of the rat PHMcc (residues 42-356) versus the corresponding bovine D M sequence. 17 Figure 1.3 Disulfide bonds in the PHM cr ystal structure. 19 Figure 1.4 Overlap of reduced and oxidized PH M crystal structures. 22 Figure 1.5 PHM active site as determined from the pre-catalytic crystal structure (1SDW). 23 Figure 1.6 PHM copper domain geometry as a function of oxidation state. 24 Figure 2.1 Structure of oleamide 48 Figure 2.2 Relationship of reactant (R) and product (P) wells for the Marcus theory for electron transfer model. 50 Figure 2.3 Reaction coordinate for a tunne ling reaction displaying the distance dependence between donor and acc eptor within the transition configuration. 55 Figure 2.4 The Dependence of the VMAX/KM for the PAM-catalyzed oxidation of the N -acylglycines on the acyl chain length. 72 Figure 2.5 The decrease in the D(VMAX/KM)app at ambient O2 as the acyl chain length increases for PAM catalysis. 73 Figure 2.6 Primary and secondary kinetic plots for N -acetylglycine and [-2H2]N -acetylglycine] 75 Figure 2.7 VMAX,app replot for N -acetylglycine and [-2H2]N -acetylglycine. 77 Figure 2.8 Replot of the D(VMAX/KM)AG vs. [O2]. 79 Figure 2.9 The effect of the micro-visc ogen (sucrose) and the macro-viscogen (Ficoll-400) on the second order rate constant (VMAX/KM). 80 Figure 2.10 Magnitude of the (VMAX/KM)OXYGEN values measured at saturating concentrations of N -acylglycine substrate at ambient oxygen concentrations. 81

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vii Figure 2.11 Converged root mean squared deviation (RMSD) values determined from equilibration of the solvated PHM structure (1SDW) for glycine extended substrates. 83 Figure 2.12 Alchemical free energy perturbation (AFEP) versus Nacylglycine chain length plot. 84 Figure 2.13 Equilibrium dynamic simulations performed for n -alkane glycine-extended substrates 85 Figure 2.14 Predicted dock ing conformations of N -acylglycines within the PHM active site. 86 Figure 3.1 Product determination by 13C NMR analysis of PAM mediated benzaldehyde imino-oxy acetic acid catalysis. 130 Figure 3.2 Determination of the PAM de pendant ratio of formed products (oxime and glyoxylate) using the im ino-oxy acetic acid substrate. 131 Figure 3.3 Stoichiometry of the enzy matic [oxygen] versus [substrate] consumption measured below th e ambient concentration of dissolved oxygen. 131 Figure 3.4 C18 -reverse phase high performan ce liquid chromatograph of (R/S)-hydroxyN -benzoylglycine, N -benzoylglycine, benzamide, benzaldehyde imino-oxy acetic acid, and benzaldoxime 133 Figure 3.5 Time course for the PAM-depe ndent conversion of 3, 2, and 0.5 mM benzaldehyde imino-oxy acetic acid to benzaldoxime (+ or -) -hydroxy-hydroxyN -benzoylglycine. 134 Figure 3.6 Resolved time course for the conversion of 6mM and 10mM -hydroxy-hydroxyN -benzoylglycine to benzamide in the presence and absence of varying [benzaldehyde imino-oxy acetic acid] 134 Figure 3.7 Replot analysis of data represented in table 2 for the D(VMAX/KM)BIAA. 137 Figure 3.8 Replot analysis of data represented in table 2 for the D(VMAX/KM)BIAA as[O2] 0uM and D(VMAX/KM)BIAA as [O2] 138

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viii Figure 3.9 The dependence of [oxygen] on D(VMAX/KM)BIAA and the [Benzaldehyde imino-oxy acetic acid] dependence on D(VMAX/KM)O2 139 Figure 3.10 QM/MM simulated reaction coor dinate for the oxidation chemistry observed for the benzaldehyde imino-oxy acetic acid C -radical oxidation with a singlet CuII-OH species. 141 Figure 3.11 QM/MM simulated reaction coor dinate for the oxidation chemistry observed for the benzaldehyde imino-oxy acetic acid C -radical oxidation with a quartet CuII-O species. 144 Figure 3.12 Combined plot of QM/MM reac tion coordinate simulation comparing the Cu-OH (singlet) and the CuII-O (quartet) spec ies again bond distance for the benzaldehyde imi no-oxy acetic acid de-alkylation event 154 Figure 4.1 Oxidative metabolites of fr om 4-phenyl-3-butenoic acid (PBA) 168 Figure 4.2 Potential energy barrier for the C C bond rotation within ethane. 173 Figure 4.3 Structures of interest for the structure-function analysis. 176 Figure 4.4 Outline of structure-function analysis used for compounds. 178 Figure 4.5 Molecular electrostatic po tential (MEP) and natural population analysis (NPA) on compounds docked into the PHM crystal structure. 194 Figure 4.6 Comparison of lone pair-ant ibond interaction for amide versus oxime moiety derived from natural bond orbital analysis. 200 Figure 4.7 Delocalization pattern as a function of oxygen insertion into N -benzoylglycine and phenylacetyla mino-acetic acid as predicted by NBO analysis 201 Figure 4.8 Delocalization pattern for carboxymethylhydroxylamine as predicted by NBO analysis. 202

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ix List of Schemes Scheme 1.1 Peptidylglycine -amidating monooxygenase (PAM) and dopamine -monooxygenase (D M) reactions. 4 Scheme 1.2 Proposed mechanism fo r lactate monooxygenase (E.C. 1.13.12.4). 7 Scheme 1.3 Reaction catalyzed by tyrosine hydroxylase. 9 Scheme 1.4 Di-oxygen activation mech anism for alkane hydroxylation by the P450 monooxyagenase. 11 Scheme 1.5 Oxidation of methane to methanol via reduced soluble methane monooxygenase. 11 Scheme 1.6 Intermediates derived from the catalytic regime of soluble methane monooxygenase. 13 Scheme 1.7 The overall reaction of ty rosinase oxidation to yield the corresponding o -quinone product catalyzed by tyrosinase (monophenol oxidase). 14 Scheme 1.8 Chemical mechanism for the hydroxylation and subsequent oxidation of tyrosine to o -quinone via tyrosinase. 15 Scheme 1.9 Postulated ki netic mechanism for PHM/D M hydroxylation contributed by Chen et al and Klinman. 33 Scheme 1.10 Postulated ki netic mechanism for PHM/D M hydroxylation contributed by Crespo et al and Yoshizawa et al 35 Scheme 2.1 Postulated pathway for the reduction of both fatty and bile Nacyl-CoA to their corresponding amide and glyoxylate products. 48 Scheme 2.2 Representative reaction for the hydrogen transfer/substrate oxidation reaction for D M and PHM 49 Scheme 2.3 Representative minimal kinetic mechanism for an ordered, sequential mechanism for substrate binding to an enzyme. 87 Scheme 3.1 Peptidylglycine -amidating monooxygenase (PAM) and dopamine -monooxygenase (D M) reactions. 106

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x Scheme 3.2 Collection of mechanisms postu lated for the hydrogen abstracting and oxidation reactions of peptidylgl ycine alpha-amidating monooxygenase and dopamine betamonooxygenase. 112 Scheme 3.3 Degenerate electronic ground state structures of the CuII-hydroperoxo and CuII-hydroxyl oxidant species proposed for direct substrat e oxidation/produc t release. 115 Scheme 3.4 Spectrophotometr ic glyoxylate analysis used in tandem with C18 RPHPLC for benzaldoxime determination. 130 Scheme 3.5 Representation of the two possible pathways for benzaldoxime and glyoxylate formation following benzaldehyde imino-oxy acetic acid oxidation in the PHM-domain. 132 Scheme 3.5b Experimental design to kinetically differentiate the two possible degradation pathwa ys for benzaldehyde imino-oxy acetic acid to its corresponding benzaldoxime product following PHM-dependent oxidation. 133 Scheme 3.6 CuM domain for the PHM active site poses for benzaldehyde imino-oxy acetic acid C -radical for the reaction coordinate corresponding to CuII-OH 142 Scheme 3.7 CuM domain for the PHM active site poses for benzaldehyde imino-oxy acetic acid C -radical for the reaction coordinate corresponding to CuII-O 145 Scheme 3.8 Minimal kinetic mechanism determined for benzaldehyde imino-oxy acetic acid oxidation as determined through primary deuterium kinetic isotope effects. 149 Scheme 3.9 De-alkylation reaction predicted for the CuII-alkoxide intermediate formation with benzaldehyde imino-oxy acetic acid as the substrate. 156 Scheme 4.1 Kinetic order for PAM and D M oxidation reactions. 164

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xi List of Spectra Spectrum 5.1: benzaldehyde imino-oxy acetic acid 1H NMR analysis (250MHz, MeODd4) and 13C NMR analysis (62.5MHz, MeODd4) 217 Spectrum 5.2: benzaldehyde imino-oxy acetic acid 1H NMR analysis (250MHz, MeODd4) and 13C NMR analysis (62.5MHz, MeODd4) 218 Spectrum 5.2: benzaldehyde imino-oxy acetic acid 1H NMR analysis (250MHz, MeODd4) and 13C NMR analysis (62.5MHz, MeODd4) 219 Spectrum 5.3: para-nitro benzaldehyde imino-oxy acetic acid 1H NMR analysis (250MHz, MeODd4) and 13C NMR analysis (62.5MHz, MeODd4) 220 Spectrum 5.4: para-methoxy benzaldehyde imino-oxy acetic acid 1H NMR analysis (250MHz, MeODd4) and 13C NMR analysis (62.5MHz, MeODd4) 221 Spectrum 5.5: para-chloro benzaldehyde imino-oxy acetic acid 1H NMR analysis (400MHz, DMSOd6) and 13C NMR analysis (100MHz, DMSOd6) 222 Spectrum 5.6: para-fluoro benzaldehyde imino-oxy acetic acid 1H NMR analysis (400MHz, DMSOd6) and 13C NMR analysis (100MHz, DMSOd6) 223 Spectrum 5.7: para-hydroxy benzaldehyde imino-oxy acetic acid 1H NMR analysis (400MHz, DMSOd6) and 13C NMR analysis (400MHz, DMSOd6) 224 Spectrum 5.8: acetophenone imino-oxy acetic acid 1H NMR analysis (250MHz, MeODd4) and 13C NMR analysis (62.5MHz, MeOD d4) 225 Spectrum 5.9: [2H2] acetophenone imino-oxy acetic acid 13C NMR analysis (62.5MHz, MeOD d4) 226 Spectrum 5.10: para-cyano acetophenone imino-oxy acetic acid 1H NMR analysis (250MHz, MeODd4) and 13C NMR analysis (62.5MHz, MeOD d4) 227

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xii Spectrum 5.11: para-nitro acetophenone imino-oxy acetic acid 1H NMR analysis (250MHz, D2Od2) and 13C NMR analysis (62.5MHz, MeODd4) 228 Spectrum 5.12: para-methoxy acetophenone imino-oxy acetic acid 1H NMR analysis (250MHz, MeODd4) and 13C NMR analysis (62.5MHz, MeODd4) 229 Spectrum 5.13: para-chloro acetophenone imino-oxy acetic acid 1H NMR analysis (250MHz, MeODd4) and 13C NMR analysis (62.5MHz, MeODd4) 230 Spectrum 5.14: para-fluoro acetophenone imino-oxy acetic acid 1H NMR analysis (62.5MHz, MeODd4) 231 Spectrum 5.15: para-hydroxy acetophenone imino-oxy acetic acid 1H NMR analysis (250MHz, MeODd4) and 13C NMR analysis (62.5MHz, MeODd4) 232 Spectrum 5.16: para-amino acetophenone imino-oxy acetic acid 13C NMR analysis (100MHz, DMSOd6) 233 Spectrum 5.17: -naphthaldehyde imino-oxy acetic acid 1H NMR analysis (400MHz, DMSOd6) and 13C NMR analysis (100MHz, DMSOd6) 234 Spectrum 5.18: [2H2]-naphthaldehyde imino-oxy acetic acid 1H NMR analysis (400MHz, DMSOd6) 235 Spectrum 5.19: para-(5-dimethylaminonapthalene-1-sulfonamido) acetophenone imino-oxy acetic acid 1H NMR analysis (400MHz, DMSOd6) and 13C NMR analysis (100MHz, DMSOd6) 236 Spectrum 5.20: [2H2]Acetylglycine 1H NMR analysis (400MHz, DMSOd6) and 13C NMR analysis (100MHz, DMSOd6) 237 Spectrum 5.21: [1H2]Propionylglycine 1H NMR analysis (400MHz, DMSOd6) and 13C NMR analysis (100MHz, DMSOd6) 238 Spectrum 5.22: [2H2]Propionylglycine 1H NMR analysis (400MHz, DMSOd6) and 13C NMR analysis (100MHz, DMSOd6) 239

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xiii Spectrum 5.23: [1H2]Butyrylglycine 1H NMR analysis (400MHz, DMSOd6) and 13C NMR analysis (100MHz, DMSOd6) 240 Spectrum 5.24: [2H2]Butyrylglycine 1H NMR analysis (400MHz, DMSOd6) and 13C NMR analysis (100MHz, DMSOd6) 241 Spectrum 5.25: [1H2]Hexanoylglycine 1H NMR analysis (400MHz, DMSOd6) and 13C NMR analysis (100MHz, DMSOd6) 242 Spectrum 5.26: [2H2]Hexanoylglycine 1H NMR analysis (400MHz, DMSOd6) and 13C NMR analysis (100MHz, DMSOd6) 243 Spectrum 5.27: [1H2]Octanoylglycine 1H NMR analysis (400MHz, DMSOd6) and 13C NMR analysis (100MHz, DMSOd6) 244 Spectrum 5.28: [2H2]Octanoylglycine 1H NMR analysis (400MHz, DMSOd6) and 13C NMR analysis (100MHz, DMSOd6) 245 Spectrum 5.29: [1H2]Decanoylglycine 1H NMR analysis (400MHz, DMSOd6) and 13C NMR analysis (100MHz, DMSOd6) 246 Spectrum 5.30: [2H2]Decanoylglycine 1H NMR analysis (400MHz, DMSOd6) and 13C NMR analysis (100MHz, DMSOd6) 247

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xiv List of Abbreviations AFEP Alchemical Free Energy Perturbation CuH Electron transfer copper CuM Oxygen activating copper CO Carbon monoxide D Deuterium (2H) Dansyl5-dimethylaminonaphthalene-1-sulfonylEPR Electron Paramagnetic Resonance EXAFS Extended X-ray absorbtion fine structure FTIR Fourier transf orm infrared spectroscopy IAA Imino-oxy acetic acid KIE Kinetic isotope effect MD Molecular dynamics MES 2-( N -morpholino)ethanesulfonic acid MEP Molecular electrostatic potential MM Molecular mechanics NBO Natural bond analysis NMR Nuclear magnetic resonance PAL Peptidylglycine amidoglycolate lyase PHM Peptidylglycine -amidating monooxygenase PHMcc Peptidylglycine -amidating monooxygenase catalytic core QM/MM Hybrid quantum mechanical/molecular mechanics QPLD Quantum polarized ligand docking

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xv Mechanistic Studies of Peptidylgl ycine alpha-Amidating Monooxygenase Neil R. McIntyre ABSTRACT Peptide hormones are responsible for cellula r functions critical to the survival of an organism. Approximately 50% of all know n peptide hormones are post-translationally modified at their C -terminus. Peptidylglycine -amidating monooxygenase (PAM) is a bi-functional enzyme which catalyzes the activation of peptide pro-hormones. PAM also functionalizes long chain N -acylglycines suggesting a potential role in signaling as their respective fatty acid am ides. As chain length increases for N acylglycines so does the catalytic efficiency. This effect was probed further by primary kinetic isotope effects and molecular dynami cs to better resolve the mechanism for improved catalytic function. The 1o KIE showed a linear decrease with increasing chain length. Neither the minimal kinetic mech anism nor the maximal rate for substrate oxidation was observed to be altered by substr ate hydrophobicity. It was concluded that KIE suppression was a function of Pre-organiz ation more effici ent degenerate wave function overlap between C-H donor and Cu(II) -superoxo acceptor with increased chain length. Substrate activation is believed to be facilitate d by a Cu(II)-superoxo complex formed at CuM. Benzaldehyde imino-oxy acetic acid undergoes non-enzymatic O dealkylation to the corresponding oxime and gl yoxylate products. This phenomena was further studied using QM/MM methodology usi ng different Cu/O species to determine which best facilitated the dealkylation event. It was determined that radical

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xvi recombination between a Cu(II)-oxyl and a substr ate radical to form an unstable copperalkoxide intermediate was best su ited to carry out this reaction. Structure-function analysis was used to rationalize the electronic features which made a variety of diverse imino-oxy acetic acid analogues such unexpectedly good PAM substrates (104-5 M-1s-1). To observe the effect oxygen insertion and placement had on substrates between N -benzoylglycine and benzaldehyde im ino-oxy acetic acid structures, PAM activity was correlated with NBO/MEP calculations on selected PHM-docked structures. This work concluded that th e imino-oxy acetic acid was a favored substrate for PAM because its oxime electronically is very similar to the amide present in glycineextended analogues.

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CHAPTER 1: Introduction The -Amidating Enzyme For some time, the amino acid sequences of peptide hormones which terminate in a -amide group have been known to be fo llowed by glycine in their prohormone sequence. For example, the sequence of -melanotropin( 1) and mellitin( 2), which terminates in valine-amide and a glutamine-amide, occurs adjacent to glycine in the precursor sequence. These observations suggested that C -terminal amide biosynthesis involved the action of a specifi c enzyme to functionalize C-te rminal glycines. In 1982, an enzyme was discovered in the porcine p ituitary which converted glycine-extended peptides to the corresponding des-glycine peptide amide( 3). Peptidylglycine -amidating monooxygenase (PAM) is a bi-functional metallooxygenase that has been the subject of deep study over a broad number of fields from physical biochemistry, metabolomics, hormonolo mics to process chemistry. High levels of PAM are found within the neurosecretory vesicles, as well as many neuronal and endocrine cells with high abunda nce in the pituitary gland ( 4, 5 ). The PAM reaction is primarily utilized in vivo for the bio-activation of peptide hormones through catalytic cleavage of a -C-N bond, truncating glycine-ultimate su bstrates to their corresponding amides. A variety of these amidated hormones function as neurotransmitters with wide distribution over the paracrine and autocrine sy stems and actually appear to be ancient physiological signal strategies ( 6). PAM also functions in the biosynthesis of amides produced from glycine-extende d fatty acid precursors( 7-10) and bile acids ( 11 ). Of these examples, the role of fatty acid amide bi osynthesis has recently been implicated in the oleamide production, which is ac tive in the sleep regulation pathway.( 7, 12, 13) 1

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CHAPTER 1: Introduction Amidation serves as a down-stream post-translational modification present within both vertebrates and invertebrates, with pos tulated contributions leading to increased ligand-receptor binding specificity for peptide and fatty acid amides compared with carboxy-terminal prototypes ( 14). PAM is the only known enzyme to catalyze substrate amidation. This suggests PAM may be the rate-limiting step for signaling peptide and fatty acid amide activation ( 6). Dopamine -monooxygenase (D M, E.C. 1.14.17.1) is a tetrameric protein (75 kDa/monomer), with both membrane and soluble forms( 15) able to catalyze the stereospecific conversion of dopamine to norepinephrine within the neurosecretory vesicles of the sympathetic nervous system( 16-18). The sympathetic nervous system consists of both neuronal axons that in teract with both smooth muscle and the catecholamine secreting cells within the adrenal medulla ( 19). Dopamine, an important neurotransmitter in the central nervous system, gains function as a neurotransmitter in the sympathetic nervous system through its in ter-conversion to norepinephrine. Catecholamine neurotransmitter function is also relevant in several neurodegenerative disease states such as Alzheimers ( 20 ) and Parkinsons (21) disease, and depression ( 22). The transformation of one well-known cat echolamine, dopamine to norepinephrine increases resulting receptor specificity. Structural Featur es of PHM and DM Peptidylglycine -amidating monooxygenase (PAM, E.C. 1. 14.17.3) is a bifunctional enzyme consisting of two hetero-domains: Peptidylglycine -hydroxylating monooxygenase (PHM) and peptidylglycine amidoglycolate lyase (PAL). Within PAM, the PHM domain has been the most extensively studied, due to its reaction and structural 2

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CHAPTER 1: Introduction homology to dopamine-monooxygenase (D M)( 17, 23). Both enzymes are structurally characterized by a solvent fille d active site separating two essential, non-coupled copper atoms( 24-26). PHM is the N -terminal fragment of bi-functional PAM. This domain is responsible for the copper-, O2-, and the ascorbate/reductant -dependent hydroxylation of the -glycyl carbon. Crystallographic evidence for PHM suggest that the two coppers are as far as ~10.6 apart ( 6, 27, 28 ) with extended X-ray absorption fine structure (EXAFS) analysis for D M in good agreement, suggesting that the coppers are greater than four angstroms apart ( 29, 30). Electron paramagnetic resonance (EPR) and EXAFS suggest that the active-site bound coppers be have completely as non-blue/type-II noncoupled mono-nuclear metal ions ( 24, 31-33). This structural a nd electronic data have extreme value, aiding mechanistic resolution when coupled with kinetic analysis for these enzymes (17, 34-36 ). The reactions for both PHM and D M involve hydrogen atom transfer from a substrate/donor complex to an activated Cu/O species, resulting in the stereospecific hydroxylation of pro-S -glycine carbon and pro-R be nzylic carbon hydroxylation (Scheme 1.1a and 1.1b). 3

PAGE 22

CHAPTER 1: Introduction Peptide O N H O HR HS OH O2H2O 2 ASC Peptide O N H O HR OH OH 2 CuIIPHM ZnII/FeIIPAL Peptide O NH2 O O OH GLYCINE-EXTENDED PEPTIDE PEPTIDE -HYDROXYGLYCINE PEPTIDE ACID AMIDE GLYOXYLATE 2SDA O2H2O 2 ASC 2 CuII2SDA NH3HO HO DOPAMINE NOREPINEPHRINE NH3HO HO HS OH D M Scheme 1.1a and 1.1b. Peptidylglycine -amidating monooxygenase (PAM) and dopamine -monooxygenase (D M) reactions. Please note, ASC represents ascorbate, SDA is semi-dehydroascorbate PHM is peptidylglycine -hydroxylating monooxygenase and PAL is peptidyl glycine amidoglycolate lyase. Peptidylglycine Amidoglycolate Lyase (PAL) In addition to the monooxygenase domain of bi-functional PAM, the PAL domain is a zinc, and recently discovered iron-depende nt enzyme which catalyzes de-alkylation of the -hydroxyglycine PHM product to its corresponding amide and glyoxylate products (scheme 1.1a) ( 37-39). In PAM, PAL is a 33 kDa monomer bound to the C terminus of PHM and is responsible carbinolamide dealkylation( 38). Precise details of the PAL mechanism have yet to be fully eluc idated, though it was broadly considered to catalyze a zinc-hydrolase-type reaction, with removal of the substrate C -hydroxyl proton by an active site base that drove th e formation of the glyoxylate aldehyde moiety ( 6, 40). It should be noted that no substrat e channeling between PHM and PAL domains has been detected ( 41 ). Therefore, it appears that -hydroxyglycine release from PHM is completely independent of subsequent PAL uptake. The iron-dependence of this reaction was unexpected and is not well understood, though mutation of a conserved 4

PAGE 23

CHAPTER 1: Introduction tyrosine residue (Tyr 564) eliminated iron binding resulting in full PAL inactivation ( 38). PAL is a ureidoglyc olate lyase (E.C. 4.3.2.3), which requires a tyrosine-bridged ZnII-FeIII complex for catalysis (38). By also utilizing calcium, PAL may actually be a unique variation of the mixed-valent di -nuclear complex detailed in purple acid phosphatase (E.C. 3.1.3.2.), making it an equally intriguing metallo-enzyme to PHM ( 4245). The bi-functionality provided by PAL represents the key difference in the reactions catalyzed by PAM and D M. Overall, this lack of bi-functionality prevents dealkylation in D M (scheme 1.1b). Though, novel substrat es have been synthesized to demonstrate this distinct chemistry for both PHM and D M. For D M, novel substrates have characterized sulfoxidation(46), epoxidation of olefins (47-49), selenoxidation( 50), and ketonization( 51 ) oxidations (phenylethanolamines( 51, 52), and -halophenylethylamines( 53-55)). Of this group, only sulf oxidation has been observed for PAM to date ( 46). Analogous to bi-functional PAM, D M catalyzes the N -dealkylation of benzylic N -substituted analogues (48), and allylic hydroxylation ( 56). The novel D M substrate analogues which demonstrate a de-alkylation process similar to that observed with the bi-functional PAM are very intriguing. In D M, N -dealkylation resulted direc tly from the monooxygenation reaction with N -phenylethylenediamine, and N -methylNphenylethylenediamine substrates( 48 ). The analogous reaction, in which only the PHM domain is utilized for bi-functional PAM have also been observe d. Two examples include; benzylglycine and benzyloxy acetic acid which were oxidized to the benzylamine and benzylalcohol species, with concomitant glyoxylat e production for both reactions (39 ). In each reaction, 5

PAGE 24

CHAPTER 1: Introduction PAL-dependent Nand Ode-alkylation reactions were facilitated through complete dependence on the PHM domain. For PHM and D M, these de-alkylation reactions provide evidence for a bond scission resulting solely from the oxidation chemistry of their monooxygenase domains. The similar ity in de-alkylation chemistry provides a potentially interesting framework to st udy oxidation mechanisms within PAM and D M catalysis (39, 46, 48, 57 ). Review of Oxygen Activation Among Monooxygenases PHM and D M are both monooxygenases, a cla ss of enzyme which activates a nucleophilic species of di-oxygen. These enzy mes have many several different routes for oxygen activation. Though to be classified as a mono oxygenase, substrate activation must catalyze di-oxygen cleavage inserting one oxygen into the product while the other is liberated as water( 58 ). Flavoproteins are known to reduce di-oxygen into hydrogen peroxide in lactate monooxygenase (E.C. 1.13.12.4) catalyzing a sequential reaction which involves an init ial reduction of the isoalloxazi ne ring oxidizing lactate to pyruvate. Oxygen facilitates the concomita nt reduction of the dihydroflavin co-enzyme and oxidation of pyruvate to acetate (Scheme 1.2). 6

PAGE 25

CHAPTER 1: Introduction HO O OO O ON N NH N CH2-(CH-OH)3-CH2-OPO3 -2 O O N H N NH H N O O CH2-(CH-OH)3-CH2-OPO3 -2 OO O ON N NH N CH2-(CH-OH)3-CH2-OPO3 -2 O O H2O Lactate Pyrvate Acetate CO2+ FMN FMNH2FMN Scheme 1.2. Proposed mechanism for lactate monooxygenase (E.C. 1.13.12.4). Co-enzyme reduction following pyruvate forma tion catalyzes formation of a flavinperoxide nucleophile which promotes pyruvate decarboxylation and water release( 58). Other flavin-dependant monooxygenases also incorporate variations of the flavinhydroperoxide intermediate into their cata lytic mechanism to activate di-oxygen, such examples are p-hydroxybenzoate hydroxylase, phenol hydroxylase, and phydroxybenzoate hydroxylase, cyclohexanone oxygenase, and bact erial luciferase( 58 ). Oxygen activation in pterin-dependent monooxygenases is very similar due, in part, to the structural homology between compounds (Fig. 1.1). Though as we will see, there are some important aspects of oxyge n activation that are no t present in flavindependent oxygen activation. 7

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CHAPTER 1: Introduction NN2NH O N N H pterin N N NH N R O O R flavin Figure 1.1. Structure comparison of flavin and pterin. Tyrosine hydroxylase is well-studied p terin-dependent monooxygenase that is important for neurotransmitter biosynthesis. The reaction involves the conversion of tyrosine to 3,4-dihydroxyphenylal anine (L-DOPA). Interestingly, this enzymes was the first observed to display the well-known [ 1,2]-NIH shift during ar omatic hydroxylation. It has recently been determined that th is intramolecular migration was dependent primarily on the degree of steric hindrance at the para-position( 59). This dependence on aromatic structure suggested that a highly re active electrophile was present. Di-oxygen activation by tyrosine hydroxylase is simila r to the flavin-peroxide of lactate monooxygenase as it involves formation of a tetrahydropterin-bound hydroperoxide intermediate. Although a pterin-hydrop eroxide species is formed, aromatic hydroxylation is dependent on ferrous iron(II)( 60, 61). The role of the iron in the catalytic mechanism is to cleave the pterin-bound pe roxide, yielding a high-spin iron(IV)-oxo species (62). This intermediate was recently trapped with tyrosine hydroxylase actually bound to the meta -position of the substrate and is responsible for hydroxylation. 8

PAGE 27

CHAPTER 1: Introduction NNH2NH O N N R NH3O O FeIIHis Glu His OH NNH2N O N H H N R O OH FeIIHis Glu His NNH2N O N H H N R OH FeIIHis Glu Hi s OO NH3O O OH O FeIVHis Glu His O H NNH2N O N H H N R OH NH3O O OH NH3O O OH RDS FeIIHis Glu Hi s NH3O O OH OH NNH2N O N H H N R OH Product Release Scheme 1.3. Reaction catalyzed by tyrosine hydroxylase. Th e electrophilic aromatic substitution of tyrosine is cat alyzed by a high-spin ferryl oxi dant, which is produced from the reduction of a pt erin-peroxide complex( 62). In tyrosine hydroxylase, the first step in oxygen activation is the two electron reduction of di-oxygen (pterin-hydroperoxide) which is required to regulate iron oxidation. Once the pterin-peroxide is formed, iron oxi dation proceeds through the concomitant formation of Fe(IV)O and 4a-hydroxytetrahydropterin. Oxid ation of the aromatic substrate is the rate limiting step (RDS) wh ich is carried out through an electrophilic aromatic substitution with the ferryl (Fe(IV)O) and tyrosine (Scheme 1.3). 9

PAGE 28

CHAPTER 1: Introduction The systems discussed illustrate an ex clusive co-enzyme dependent activation of oxygen with the flavins while the pterin co -enzyme containing system was used to activate a high-spin ferryl complex. The next monooxygenase demonstrates di-oxygen activation with the heme-dependent cy tochrome P450 monooxygenases. The P450 monooxygenases are a very well-studied superfam ily of enzymes. The mechanism of dioxygen activation by an iron co-factor involve s the modification of the heme-bound ferric (Fe+3) spin state from low to high( 58 ). Substrate binding results in the displacement of an axial water ligand lowering the coordination numb er from six to five. Prior to oxygen activation, a single elec tron transfer event from an exogenous reductant results in a heme-bound ferrous species. Di-oxygen bind ing results in a low-spin peroxy radical which converted to a heme-peroxide moie ty through sequential electron and proton transfer events. The ferryl-oxo (FeIV=O) species is formed th rough protonation of the heme-peroxide. This is a similar activated oxygen species to that observed with tyrosine hydroxylase, though the activate r oute is drastically differe nt. Substrate oxidation follows through a concerted rebound mech anism in which C-H cleavage and C-OH oxidation occur in a single step (Scheme 1.4). 10

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CHAPTER 1: Introduction N NN N FeIII LS Cys OH2 N NN N FeIII HS Cys N NN N FeIII Cys O N NN N FeIV Cys O N NN N FeIII LS Cys O R-H e-N NN N FeII Cys R-H R -H O2O e-, H+ R-H R-H e-, H+ H2O R H R-OH H2O Scheme 1.4. Di-oxygen activation mechanism for alkane hydroxylation by the P450 monooxygenase. Di-oxygen activation can also occur with non -heme di-iron scaffolds such as soluble methane monooxygenase (sMMO; E. C. 1.14.13.25). Present in methanotropic bacteria, sMMO catalyzes the oxidation of methane to methanol (Scheme 1.5). CH4 O2NAD(P)H H+ sMMO CH3 HO NAD(P)+ H2O Scheme 1.5. Oxidation of methane to methanol via reduced soluble methane monooxygenase. This is an essential transformation as me thanol is further oxidized to formaldehyde eventually with energy stored as NADH, re spectively. This reaction is carried out through a three subunit scaffold that includes a regulatory subunit which is required for activity (MMOB). A reductase subunit (MMOR) is also present to s huttle electrons from NADH to the monooxygenase active site (MMOH)(63, 64). In the resting state (Hox) 11

PAGE 30

CHAPTER 1: Introduction the positive charge of the two high spin ferric (FeIII) atoms are countered by two glutamate and bridgi ng hydroxide ligands( 65). The octahedral ge ometry of the iron is completed through the coordination of two a dditional histidine residues. Upon reduction of Hox to FeII/(Hred), the MMOH subunit loses -bis-hydroxyl character causing an ironbound glutamate to undertake a carboxylate rearrangement from a free carboxy-oxygen atom to a bridging position between the FeII-FeII. This step causes a shortening of the diiron interatomic distance slightly allowi ng the MMOH di-ferrous iron to rapidly and irreversibly bind di-oxygen to yield intermediate O ( 66 ). Di-oxygen then directly complexes with the di-iron cluster caus ing the oxidation of each iron to the -1,2-peroxodiferric intermediate, P*( 66). Donation of a proton modifies P* to a di-ferrichydroperoxo species containing a -( -peroxide core structure which is designated species P ( 67 ). A subsequent proton results in di-oxygen cleav age leaving water and a FeIV-FeIV bis-oxo species ( Q ), the most powerful oxidizing species in nature ( 66). The conversion of P occurs without net change in the oxidation state of the iron core as shown spectroscopically with Mssbauer displaying low isomer shifts indicative of FeIV( 68). The decay of Q was found to be directly proportional to substrate concentration represented by a la rge kinetic isot ope effect on Q decay of 50 ( 66). Q decay occurs in two sequential steps: substrate binding (species QS ) and oxygen transfer to form bound product (species T ). The addition of substrate to the species Q (yielding species QS ) is characterized by the FeIV-FeIV bis-oxo intermediate which abstracts substrate hydrogen to give a substrate radica l and carbocationic species with an adjacent mixed valent FeIII-FeIV-OH intermediate (species R ). The mechanism of hydroxylation has not yet been fully elucidated, though a rebound mechanism involving direct substrate 12

PAGE 31

CHAPTER 1: Introduction hydroxylation of the enzyme-bound radical or substrate carbocation quenching mechanism facilitated by the FeIII-OH moiety of species R has been postulated( 58). The resulting substrate oxidation a nd alcohol release allow the FeIII-FeIII bis-hydroxyl species of Hox to be restored (Scheme 1.6). FeIIIH O FeIII 2NAD(P)H + H+2NAD(P)+ FeIIO FeII O2OO FeIIIFeIIIO H2O FeIVO FeIV O S-H FeIIIO FeIII HO S S-OH O H HOXHREDO P*Q T O FeIIO FeII O HOH HOH HOH HOH O FeIIIFeIIIO H P HO O FeIVO FeIV O QS S-H FeIIIH O FeIV O R S /S+ FeIIIFeIIIO HO-S Soluble Methane Monooxygenase (E.C. 1.14.13.25) MMOR B B B B B B B B B B Scheme 1.6. Intermediates derived from the catalytic regime of soluble methane monooxygenase. 13

PAGE 32

CHAPTER 1: Introduction The role of the copper co-factor for monooxygenase di-oxygen reduction is determined in binuclear proteins as being either magnetically coupled or non-coupled For the coupled binuclear copper monooxygena ses, monophenol oxidase (tyrosinase; E.C. 1.14.18.1) is an ideal example of an anti-ferr omagnetically coupled di-copper monooxygenase. Tyrosinase is an essentia l copper-containing enzyme present in organisms regulating pigmentation through the production of melanin( 69, 70). Present in melanocytes, this essential enzyme cataly zes the hydroxylation of monophenols to orthodiphenols and the subsequent two electron oxidation to orthoquinones with molecular oxygen( 69, 70) (Scheme 1.7). Melanin is a ubiquit ous polymer composed of monomeric precursors formed from the intramolecular cyclization of aminoalkyl substituted oquinones which contain the necessary indole character for melanogenesis( 71). OH O2 O O H2O Tyrosinase E.C. 1.14.18.1 Scheme 1.7. The overall reaction of tyrosinase oxidation to yiel d the corresponding oquinone product catalyzed by tyrosinase (monophenol oxidase). Recently, the X-ray crystal structure of ty rosinase was solved by Matoba et al.(72 ) providing many novel insights into th e chemical mechanism. The oxy (Scheme 1.8, blue box) form, 2: 2 side-on peroxide bridge, is consistent geom etrically with type 3 copper proteins such as catec hol oxidase and hemocyanin( 70, 73-75). Through flash photolysis, the binding and reduction (2e-) of di-oxygen by deoxy tyrosinase (scheme 1.8, green box) was shown to be rate limiting for phenol oxida tion through high enthalpic 14

PAGE 33

CHAPTER 1: Introduction barriers( 76). This thermodynamic barrier was sugge sted to assist secondary structure dynamics required which accommodate the shorte r inter-copper distance characteristic within the 2: 2 side-on geometry. Substrate orientation to the oxy state is stabilized through a hydrophobic interaction with a histidine residue from CuB orienting the phenolic C-O toward CuA through a stacking interaction ( 69 ) (Scheme 1.8). CuA IIO CuB II O NHIS NHIS NHIS NHIS NHIS NHIS O CuA IIO CuB II O NHIS NHIS NHIS NHIS NHIS NHIS O H CuA IIO CuB II O NHIS NHIS NHIS NHIS NHIS NHIS O H CuA ICuB INHIS NHIS NHIS NHIS NHIS NHIS O2CuA IIO CuB II O NHIS NHIS NHIS NHIS NHIS NHIS H+H2O O O H+ OH CuA IIO CuB II O H NHIS NHIS NHIS NHIS NHIS NHIS O Scheme 1.8. Chemical mechanism for the hydr oxylation and subsequent oxidation of tyrosine to o -quinone via tyrosinase. The proximity of the ortho -phenolic hydrogen to the side -on coordinated peroxo group facilitates electrophilic attack of Cu2O2 moiety on the aromatic ring( 69). This results in an activated substrate bridging geometry that involves a phenolic C-O coordinated directly to CuB in addition to a phenoxo bridge between CuA and CuB. This bi-dentate diphenolic intermediate further modifies the original oxy 2: 2 side-on geometry by decreasing the di-oxo interaction allowing the 2 oxygen to be stabilized by the ortho phenolic proton which migrates to form an 2 hydroxyl moiety. This interaction is believed to allow rotation of the O-O axis within the peroxo ligand towards the phenol 15

PAGE 34

CHAPTER 1: Introduction whereby concomitant cleavage of the O-O bond occurs. The o-quinone product is released with a water molecule though pr otonation of the bi-dentate di-phenolic intermediate leaving the deoxy tyrosinase form (scheme 1.8, green box)( 69 ). Non-coupled monooxygenases The focus of the following section will bui ld on the nature of di-oxygen activation and discuss in detail the magnetically non-coupled di-copper monooxygenases. As with all oxygen activating enzymes, the use of a non-coupled di-copp er active site to activate di-oxygen presents unique chemistry. With in this field, both peptidylglycine amidating monooxygenase (PAM) and dopamine -monooxygenase (D M) are part of a unique class of type-II copper enzymes. Seve ral proteins are cate gorized within this area: PHM, D M, monooxygenase X (MOX)( 77), tyramine -monooxygenase (T M)( 78 ), and dopamine -hydroxylase-like (D HL)(79). Sequence Homology between PHM and DM Shown in Fig. 1.2, is the aligned amino acid sequence of the PHM catalytic core (PHMcc) evaluated with respect to bovine D M. Conserved residues are shown in the consensus sequence with an exclamation point (!) while similar residues are denoted with an asterisk (*). Results revealed a significant 28% sequence identity and a 40% sequence similarity extending throughout the catalytic domain comprised of approximately 270 residues(6, 23 ). The corresponding active site residue identification numbers are listed for clarity in Table 1.1, respectively 16

PAGE 35

CHAPTER 1: Introduction Figure 1.2. Consensus sequence of the rat PHMcc (residues 42-356) versus the corresponding bovine D M sequence. Please see text for more detail. Dopamine Peptidylglycine -monooxygenase -hydroxylating monooxygenase Histidine 265 Histidine107 CuHHistidine266 Histidine108 Histidine336 Histidine172 Tyrosine233 Tyrosine79 Glutamine170 Glutamic Acid268 Histidine415 Histidine242 CuMHistidine417 Histidine244 Methionine490 Methionine314 Tyrosine494 Tyrosine318 Table 1.1. Sequence homology comparing active site residues from PHM to analogous residues in D M. 17

PAGE 36

CHAPTER 1: Introduction The role of disulfide bonds in the PHM structure are currently unresolved, though their position (Fig. 1.3) suggest that they may aid in defi nition of each respective copper domain. Further evidence for the structural definition within the tertiary structure of PHM provided by conserved disulfide bonds comes from the ease at which enzyme bound coppers may be removed from thei r respective binding domains without denaturation (24). This suggests that copper is not necessary for the folding of either PHM or D M within the endopla smic reticulum (ER) and ma y be transported to the protein once the folded structure is establis hed to yield the fully active structure. Evidence for copper chaperones includes HAH1, which transports copper to both Menkes and Wilson proteins( 80-82 ). Both proteins are coppertranslocating P-type ATPases found in the trans-golgi apparatus which may function to deliver copper to subsequent enzymes (ATP7A) for delivery of c opper to dopamine beta-monooxygenase and peptidylglycine alpha-amidating monooxygenase within the trans-golgi network. Therefore, experimental evidence supports the sequential process of protein folding followed by copper binding for PHM and D M. 18

PAGE 37

CHAPTER 1: Introduction CuMCuHCys227 Cys334 Cys315 Cys293 Cys81 Cys47 Cys114 Cys131 Cys126 Cys186 CuMCuHCys227 Cys334 Cys315 Cys293 Cys81 Cys47 Cys114 Cys131 Cys126 Cys186 Figure 1.3. Disulfide bonds in the PHM crystal st ructure in relation to filled copper domains. Substrate Specificity for PAM Among Glycine-Extended Peptides As shown in Fig. 1.5, the C -terminal glycine-termini is utilized for substrate positioning in PHM. Beyond glycine-extended substrates, D-alanine-extended substrates have also been observed to undergo substrate oxidation( 83 ). Though in the position adjacent to the C -terminus glycine, Tamburini studied structure-activity relationship of peptide substrates for PAM. Using the substrate N -dansyl-(Gly)4-X-Gly, where X 19

PAGE 38

CHAPTER 1: Introduction represents the amino acid mutation for each of the twenty natural amino acids, amidation activity was evaluated. Overall, the identity of the amino acid does not affect the ability of PAM to amidate, though hydrophobic residues (X = phenylalanine and tyrosine) are preferred ( 84, 85 ). Electron Transfer between CuH and CuM from X-Ray Crystallography Extensive research has been reported re garding the pathway fo r electron transfer between copper sites and the nature of O2 activation in D M and PHM( 28, 86). Comparison of the structures of PHMoxidized with and without bound peptide reveal no significant differences in structure( 28 ). The coordination of the two active site coppers is similar in both the oxidized and reduced structures. The CuH remains in a planar T-shaped coordination geometry, coordinated by three histidine ligands (H107, H108, H172). The Cu-N bond lengths for H107 and H108 (1.9-2.2 ) are slightly shorter than H172 (2.1-2.6 ), which decrease by 0.4 in the reduced structure. The CuM domain remains tetrahedral, coordinate d in both oxidized and reduced forms by H242, H242, M314 and water. Overlay of CuH and CuM sites from oxidized and reduced PHMcc highlights the si milarities between the two structures (Fig. 1.4). The two greatest differences are a change in th e position of the water molecule bound to the CuH and the tighter ligation by H172 in the reduced structure. These similarities between oxidized and reduced structures were thought to promote electron tr ansfer between the sites by reducing reorganization energy( 28). PHM Structure The shape of the oxidized PHM struct ure is composed of two 9-stranded sandwich domains that are roughly equivalent in size, defined as a prolate ellipsoid ( 87 ). 20

PAGE 39

CHAPTER 1: Introduction The inter-domain surface is bridged by a 500 2, solvent accessible in terface. There is one copper per -domain, each defined by a unique ligand set. Each domain is centered around their respective copper at om. One copper center (CuM) has two N -histidines ligands (His240 and His242) and a methionine sulfur ligand (Met314) (Fig. 1.5).The CuH center, has three N -histidine ligands (His107, His108, His172). Crystal structures for substrate bound PHM have been established for the oxidized E-2Cu(II)( 87), the reduced E-2Cu(I)(28 ), and pre-catalytic (E-2Cu(I)-O2) enzyme forms( 27). The crystal structure with bound substrate shows its bi nding site close to the CuM site where O2 binding and activation are thought to occur. As shown in Fig. 1.4, there is no large structural shifts between redox states of substrate bound PHM. This de monstrated closure of the domains was unlikely. Within this figure, th e substrate binding mo tifs are also shown, with the substrate N -acetyl-3,5-diiodotyrosylglycine displaying a salt-bridge to the guanidine group of arginine 240 of the carboxy terminus of the glycine-extended substrate (Fig. 1.4). The circled -glycyl carbon within this figure contains the proS hydrogen which is abstracted via the c opper-superoxo nucleophile. Resolution of the PHMcc (residues 42-356) displays the interatomic distance between CuM and CuH to be ~11 which does not appear to change as a function of substrate binding or oxidation state. 21

PAGE 40

CHAPTER 1: Introduction Figure 1.4. Overlap of reduced ( red ) and oxidized (blue ) PHM crystal structures. 22

PAGE 41

CHAPTER 1: Introduction Figure 1.5. PHM active site determined from the pre-catalytic crystal structure (1SDW). Please note, that th e endogenous IYT structure was replaced by IYG as binding orientations were super-im posable. Circled is the C -glycyl atom. Copper Domain Geometry Revisited The copper geometry was determined from the X-ray structure to change as a function of oxidation state within their domains. In the oxidized PHM structure, CuM is square pyramidal and CuH is square planar, wh ile in the reduced form CuM is tetrahedral and CuH is T-shaped (Fig. 1.6). Though, EXAFS has determined more precise bond lengths which show the oxidized geometry of CuM is more of a dist orted tetrahedral due to the absence of Met314 interaction( 33). Upon reduction, shortening of the Cu-SMet314 bond length by over 0.3 well-describes the shown te trahedral geometry. For CuH, EXAFS data showed that only two of the thr ee histidines were coor dinated by the copper, detailing a weaker bonding intera ction for one of the histidines resulting instead in a two coordinated reduced form. As we will learn, these slight discrepancies between the X23

PAGE 42

CHAPTER 1: Introduction ray and EXAFS data are the initial cause of division within events following the reductive phase of the reacti on. Specifically, the crystal structure copper geometries would support a substrate mediat ed electron transfer event that is very fast, while the EXAFS would predict slow electron transfer due to the increased degree reduction alters the copper domains ligand environment( 88). One postulated source of error for the differing spectroscopic conclusions may have arisen from a photoreduction event from the X-ray beam causing the solved crystal structure to be an av erage of reduced and oxidized states( 88). CuM IIHis242 His244 OH2 OH2 Met314 Met314CuM I His244 His242 OH2 CuH IIH2O His108 His172 His107 His108CuH I His107 His172 1e-1eH2O H2O Oxidized CuM Domain Reduced CuM Domain Oxidized CuH Domain Reduced CuH Domain Figure 1.6. PHM copper domain geometry as a function of oxidation state. 24

PAGE 43

CHAPTER 1: Introduction Background Postulated Mechanisms For both PHM and D M, the first step of the catalytic cycle is initiated by the reduction of each enzyme-bound copper by an exogenous reductant( 6, 17); in vivo studies suggest it is ascorbic acid( 1, 89). For reduction, a ping-pong mechanism is utilized such that the reductant irreversibly redu ces each copper site allowing di-oxygen and substrate interaction only with the reduced form of the en zyme. This kinetic trend suggests that once copper reduction occurs, the affinity of the enzyme for the oxidized molecule is low with release occurring much faster than subsequent substrate binding steps. Initially performed to conclusively determine the ab sence of a coupled bi-nuclear site, Brenner et al. used burst phase kinetics to de monstrate that pre-reduced coppers were catalytically competent to hydroxylate the substrate ( 90, 91 ). Furthermore, a presteady state burst was observed which was eq ual in amplitude (1 product : 1 enzyme equivalents) to the enzyme concentration, i ndicating that the rate of product release was slow with respect to the chemical reaction( 92). This allowed a concomitant copper reoxidation with product release to be observed, thus showing that the reductant and substrates show preference for diffe rent redox states of the enzyme. The complexity within the chemical mechanism for both PHM and D M has been deduced primarily from kinetic isotope effects (KIE). Kinetic isotope effects (KIEs) are incredibly versatile tool for mechanism an d transition-state structure elucidation in enzymatic reactions ( 93, 94 ). The presence of KIEs correspond to a change in bonding force constants in the molecule of interest( 95, 96). As defined under the BornOppenheimer approximation, the uniqueness of this probe is based on the unaltered 25

PAGE 44

CHAPTER 1: Introduction potential energy surface of molecule s containing isotopic substitution, ( 95-97). The non-perturbing nature of is otopic labeling allows changes in bonding and structure to be observed directly from the magnitude of KIEs. This correlates directly with changes in vibrational phenomenon between reactant and tr ansition states. Kinetic isotope effects are determined from the product of several terms (equation 1). The first term is the mass, moment of inertia (MMI), followed by zero point energy (ZPE), and excited state energy (EXC). K IE = MMI ZPE *EXC Equation 1.1 The transition state is defined as a purely dynamic and transient theoretical vibrational state predicted along a reaction coordinate, estimated to last 10-13 s-1(less than a single bond vibration)(98-103 ). Thus, the transition state becomes a theoretical treatment for the energetic maximum of a reaction coordinate to associate the conversion of a purely vibrational event to a translatio nal one. Thus, the transition state can be considered the maximum potential energy observed on the surface of a reaction coordinate with an imaginary frequency in addition to normal vi brational modes (3N7)( 94, 97). The imaginary frequency actually f unctions in the decomposition of this activated complex. A stable frequency at th e transition state would yield a reactantor product-like intermediate that may not favor spontaneous decomposition once the activation energy requirement for reaction was reached. Kinetic isotope effects can be studied on steady-state parameters defined by initial rate kinetics. For a bi-reactant system, these parameters include KM,A, KM,B,VMAX, and VMAX/KM,A, and VMAX/KM,B. The KM terms are referred to as the Michaelis constant, 26

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CHAPTER 1: Introduction each substrate has their individually defined KM value denoted with either an A or B. The kinetic significance of the Michaelis constant is derived from the substrate concentration (either A or B) that is requir ed to reach the half maximal rate for the reaction. KM is essentially a measure of the affin ity an enzyme has for a particular substrate, and is defined as the ratio of enzyme-substrate (E-S) complexes which undergo rate-determining chemistry versus substrate re lease from the E-S complex. This maximal rate, referred to as VMAX differs from the Michaelis consta nt as it is not dependent on the substrate (either A or B) as it only de fines reaction rates observed during substrate saturation. VMAX can be defined as the rate of product release from productively bound substrates. The VMAX/KM term is defined as the sec ond-order rate constant which includes all steps involved in the addition of ei ther substrate A or B, up to and including the first irreversible step. A more common interpretation of VMAX/KM values is that it represents the catalytic efficiency of a part icular substrate, binding to the enzyme such that the release of product is observed. Th e phenomenon of substrate saturation with a bi-reactant reaction can be furt her defined as apparent KM,VMAX, and VMAX/KM values. These terms describe the relationship of reacti on velocity when one s ubstrate is saturating while the other is below this concentration. The observed trend in apparent kinetic parameters as a function of varying substrat e concentration will later be shown to give valuable insight into enzyme mechanism when coupled to KIEs. KIE nomenclature usually looks at the effect isotopic substituti on on product release and catalytic efficiency as the relationship between LIGHTVMAX/ HEAVYVMAX and LIGHTVMAX/KM/ HEAVYVMAX/KM. 27

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CHAPTER 1: Introduction Minimal Kinetic Mechanism Analysis of a minimal kinetic mechanism of an enzyme allows the researcher to understand the order of substrate addition to the enzyme and the nature of product release. This is done by examining variab les which affect kinetic rates of the enzymatic reaction. For example, one usually begins by varying substrate concentrations. A fundamental assumpti on about enzymes is that once substrate saturation is achieved, catalysis must be enti rely rate-determining. This is defined by the hyperbolic trend observed with well-behave d Michaelis-Menton kinetics. The kinetic mechanism allows the nature of E-S and E-P complexes to be established, thereby providing insight into th e dynamic architecture within the active site. Role of Tunneling in C-H Bond Cleavage The Klinman group has demonstrated a temperature dependence on both the primary and secondary intrinsic isotope effects for substrate C -H bond cleavage catalyzed by PAM. The obser ved non-classical Arrhenius behavior on the intrinsic isotope effect suggest that the probability of hydrogen atom transfer is by dominated quantum mechanical tunneling( 104). This result was unique when compared with better understood systems exhibiting this phenomenon (example soybean lipoxygenase ( 105, 106)). Due to the high degree of solvent acces sibility within the PHM active site the contribution from environmental reorganizati on energy and protein vi bration suggest that hydrogen tunneling would be an unlikely primary catalytic strategy. The reorganizational energy is required to contro l the energy to attain degenerate states 28

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CHAPTER 1: Introduction between reactant and product while protein fr equency modulates the distance between the C-H donor and the Cu/O2 acceptor. Computational Studies The first density functional theory (DFT) computational analysis of the PHM/D M reaction coordinate was used by Chen and Solomons (scheme 1.9)( 107, 108). This study used a truncated model of th e PHM active site to probe the reaction thermodynamics for C -H bond cleavage in formylglycine. Their results suggested that side-on ( 2) copper(II)-superoxo was a more favor able nucleophile compared to the 1copper(II)-hydroperoxo for C-H bond cleavage (14 versus 37 kcal/mole). Utilizing frontier molecular orbital analysis (FMO), the 2-copper(II)-superoxo wa s rationalized as increasing the availability of oxygen or bital overlap toward the hydrogen donor. According to their calculati ons, electron transfer from CuH was predicted to follow C -H bond cleavage. Subsequent theoretical studies utili zed hybrid quantum mechanical/molecular mechanics (QM/MM) calculations with D M and PHM to postulate a high-valent copper-oxo nucleophile responsible for CH bond cleavage, a hydrogen acceptor based on the mechanistic observations cytochrome P450( 109-111). This mechanism puts forth, instead of a copper-superoxo species, a highly reduced copperIII oxyl species for substrate C H bond cleavage. For these species to exist, electron tran sfer must occur prior to C-H bond activation. Both studies which conclude d the high-valent copper-oxo species are very controversial ( 112, 113). As mentioned earlie r di-oxygen reduction has strong support coupling it to substrate C-H ab straction. This would eliminate the possibility that a copper(III)oxo nucleophile is present in the reaction coordinate of 29

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CHAPTER 1: Introduction either PHM or D M. Though, further discussion is warranted as these are computationally elegant studies that offer insight the unique function of these systems while simultaneously offering potential mechanisms ( albeit unintentionally ) to improve the catalytic power of these systems and model systems therein associated with C-H bond reduction. Therefore, these mechanisms provide information about the upper limits possible for the catalytic power of PHM and D M. Evidence of CuII-superoxo Intermediate Since the hydrogen abstraction mechanism is governed by quantum mechanical tunneling, the 18O kinetic isotope effect s performed on PHM and D M only offer mechanistic information up to and incl uding the O-O cleavage step, respectively( 113 ). These are informative probes which can be coupled with C H/D kinetic isotope effects to postulate the hydrogen ab stracting species, though thes e heavy atom effects are independent of substrate oxidation and product release steps. For both PHM ( 35, 114) and D M ( 115, 116), the dependence 18O KIE upon substrate deuteration show that C-H bond cleavage precedes the O-O required for substrate hydroxylation, yielding a Cu(II)hydroperoxo and a substrate radical. Sp ectroscopic data using rapid freeze quench methodology further suggested that an EPRsilent copper-superoxo species was formed as O-O and C-H cleavage are tightly coupled ( 117 ). Unfortunately during the substrate oxidation step, the differentiation between each postulated mechanism becomes difficult to isolate and distinguish in the absen ce of a suitable reaction probe. Evidence for a Radical Substrate Intermediate The bulk of work in support of a substr ate-free radical within this area comes from the D M literature. There have been seve ral studies which built substantial 30

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CHAPTER 1: Introduction evidence for a substrate radical mechanism during turnover ( 118-121 ). The first approach used substrate-based inhibitors to probe electronic effects to define the degree of polarization within the transi tion state. As this class of inhibitors requires C-H bond cleavage to occur to facilitate inactivati on, measurement of the partition ratio as a function of substrate deuteration defined the pop ulation which went eith er to oxidation or inactivation product. It was observed that there was an isotope effect for C-H bond cleavage as a function of oxygen concentration, though not on th e partition ratio directly. From this, a sequential reaction which broke a C-H bond then oxidized substrate by way of a stable substrate radi cal was supported. Further evidence for this sequential mechanism was determined from the aromatization of 1-(2-aminoethyl)-1,4cyclohexadiene to form a phenylethylam ine product that was inert to oxygen incorporation(122). Postulated Reaction Mechanism for PHM/DM The following mechanisms display a c opper-superoxo nucleophile for substrate activation (Scheme 1.9). These mechanisms diffe r in the initial coordination geometry of the di-oxygen species to copper as either side-on or end-on. The Solomon group spectroscopically va lidated a side-on/ 2 species from bio-mimetic studies( 123, 124 ). The side-on copperII-superoxo species was shown to be more thermodynamically favorable compared with the end-on/ 1 analogue from hybrid QM/MM calculation ( 107, 108). Recently, evidence for the end-on/ 1 species has been obser ved in the PHM crystal structure (1.85 ) warranting further study of this species as the hydrogen acceptor ( 125 ). Both side-on and end-on mechanisms assimilate as an end-on/ 1 CuII-hydroperoxo species is predicted following C -H bond cleavage. The mechanism put forth by the 31

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CHAPTER 1: Introduction Solomon group predicts direct C -substrate radical hydroxyla tion, followed by a radical recombination event resulting in the simu ltaneous reduction of the copper-hydroperoxo species and hydroxylated product release. The remaining CuII-oxyl radical species provides the necessary driving force for in tramolecular electron transfer from the CuH site to traverse 11 completing the reaction. Conversely, the CuII-hydroperoxo species in the mechanism postulated by Klinman is reduced by a intra-molecular electron transfer event from the CuH site yielding a CuII O radical via homolysis. Radical recombination of substrate and Cu/O radical sp ecies result in a covalent, inner-sphere alcohol intermediate. Product release is facilitated by hydrolysis of this intermediate. 32

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CHAPTER 1: Introduction R O N H O OH H H CuIICuIIMet His His His His His i) 2eii) O 2CuIICuIMet His His His His HisO O Electron transfer Follows CH cleavageR O N H O OH H CuIICuIMet His His His His HisO OH R O N H O OH H CuIICuIMet His His His His HisO R O N H O OH H CuIICuIMet His His His His HisO OH OH 1 e-R O N H O OH H CuIICuIIMet His His His His HisO R O N H O OH H CuIICuIIMet His His His His HisO R O N H O OH H CuIIMet His His His His HisOH 1 e-R O N H O OH H CuIICuIIMet His His His His HisOH OH R O N H O OH H CuIICuIMet His His His His HisO OH Inner Sphere Alcohol Mechanism H H2O Cu/O2 reduction and C -H bond oxidation are SH2ODirect OH Transfer Mechanism Product Re lease Product Release R O N H O OH H CuIICuIMet His His His His HisO OH CuIICuIMet His His His His HisO O Copper-superoxo AcceptorR O N H O OH H H Copper-superoxo Acceptor CuIICuIMet His His His His HisH2O R O N H O OH H O OH CuIICuIMet His His His His HisH2O R O N H O OH H O OH H2O H2O H+, H2OOH2 Electron Transfer Electron Transfer CuIIOH2 tightly coupled. a l i e n t M e c h a n i s t r i c F e a t u r e s Scheme 1.9. Postulated kinetic mechanism for PHM/D M hydroxylation contributed by Chen et al ( left ) and Klinman ( ri g ht ) 33

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CHAPTER 1: Introduction Two entirely theoretical mechanisms put fort h (Scheme 1.10), proposin g that instead of a copper-superoxo species, a highly reduced copper oxo is utilized for substrate C H bond cleavage. This mechanism implies the un-coupling of di-oxygen reduction from substrate activation, analogous to the ferryl oxidant (FeIV=O) (109, 113). Through hybrid QM/MM simulations, the CuIII oxide/CuII oxyl species were determined to be thermodynamically favored versus CuII-superoxo and -hydroperoxo species( 109-111). These mechanisms propose an environmenta l modification of the initial end-on/ 1 CuII superoxo species through coupling of an intr a-molecular electron transfer to the sequential acquisition of two protons to release a water molecule resulting in a copper oxyl species. The simulation performed by Crespo et al is by far the most computationally elegant simulation of the theoretical models introduced ( 109). Here, a CuII-oxyl species was predicted to exist with two unpaired electrons ferromagnetically coupled to an unpaired electr on delocalized within the CuM ligand domain. With a quartet spin ground state of th is complex, the H-abstractio n event was assumed to be concerted with hydroxylation of the substrate radical, with a spin inversion event from quartet to doublet ground state accompanying substrate oxidation. This event represents simultaneous substrate oxidation and hydroxylated product rel ease, leaving the L3CuII to bind a water molecule to restore distorted te trahedral geometry in the oxidized, resting state. The adjacent mechanism proposed in Scheme 1.10 by Yoshiwaza et al. determined that a triplet ground state is mo st thermodynamically favorable species for Habstraction by a CuII oxyl species ( 110, 111). The following substrate oxidation reaction undergoes a spin inversion to yield an antifer romagnetically coupled si nglet state to drive concerted H-abstraction with product release. 34

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CHAPTER 1: Introduction Electron transfer Precedes C-H cleavage SR O N H O OH H H CuIICuIIMet His His His His His i) 2eii) O 2CuIICuIMet His His His His HisO O R O N H O OH H H CuIICuIIMet His His His His HisO O H R O N H O OH H H CuIICuIIMet His His His His HisO OH R O N H O OH H H CuIICuIIMet His His His His HisO H H2O R O N H O OH H CuIICuIIMet His His His His HisOH H-Abstraction +2 +2 R O N H O OH H CuIICuIIMet His His His His HisOH2 Hydroxylation+2 H2O OH Electron Transfer R O N H O OH H H CuIICuIIMet His His His His HisO R O N H O OH H CuICuIIMet His His His His HisOH H-Abstraction R O N H O OH H CuIICuIIMet His His His His HisHydroxylation H2O OH Doublet Species Singlet Species OH2 a l i e n t M e c h a n i s t i c F e a t u r e s : Cu/O2 reduction and C -H bond oxidation are entirely uncoupled. eScheme 1.10. Postulated kinetic mechanism for PHM/D M hydroxylation contributed by Crespo et al .(left) and Yoshizawa et al .(right) 35

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CHAPTER 1: Introduction Beyond the mechanisms proposed in Schemes 1.9 and 1.10 for PHM/D M substrate hydroxylation, a fifth mechanism was omitted. This mechanism was referred to as the superoxide channeling mechanism and builds on so me challenging spectroscopy to postulate this chemi cally innovative hypothesis( 86). Binding trends of copper monoxide (CO) binding to the reduced PHM structure showed a 0.5 CO : CuI stoichiometry, with the coppe r-carbonyl signal (FTIR stretch) occurring solely at CuM, respectively. Interestingly, in the presence of an enzyme bound substrate; the coppercarbonyl signal migrated completely to the CuH domain. As the presence of substrate was not found to perturb the geometry of either copper domain( 88), it was reconciled that the reduced CuH domain must function to bind dioxygen yielding a one electron reduced superoxide which traverses the 11 active site to CuM I, binding as a CuM II-superoxo complex for C -H bond activation. Therefore th is mechanism fundamentally differs from each listed above as oxygen activation oc curs with a concomitant electron transfer event from CuH. Unfortunately, the body of eviden ce does not support this hypothesis as there has never been any detectable superoxide /hydroperoxide species to leak out of the active site and all oxygen activ ation steps are always coupled with hydroxylated-product release. Introduction to Dissertation Chapters This dissertation will first look at the quantum mechanical aspect of C -H bond cleavage by observing the primary kinetic isotope effects for N -acylglycines as a function of chain length. A linear decrease in the magnitude of the observed KIE was interpreted using both molecular mechanics (MM) doc king simulations as well as molecular dynamics simulation called Alchemical Free Energy Perturbation (AFEP). AFEP allows 36

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CHAPTER 1: Introduction the relative energy of bound substrates to be directly compared. This study concluded that the decrease in the KIEobserved for these substrates involves a structure-dependent increase in pre-organization which serves to decrease the conf ormational sampling of the longer substrates allowing more efficien t wave function overlap to be present upon substrate binding. The next chapter involves the rationa l design of a substrate to deduce the oxidation species involved in substrate hyd roxylation. Benzaldehyde imino-oxy acetic acid was determined to undergo a PAL-inde pendent, non-enzymatic dealkylation reaction following PHM oxidation to benzaldehyde oxime and glyoxylate products. Therefore, this substrate was well adapted to st udy the monooxygenase domain of bi-functional PAM, the PHM-domain, independently of the PAL-dependent dealkylation observed with glycine-extended substr ates. A hybrid quantum mech anical-molecular mechanical simulation was performed to model this non-en zymatic de-alkylation to determine which oxidation species in PHM was responsible for substrate oxidation. The results suggest that a covalent intermediate which follo ws radical recombination between a copperII-oxyl and a substrate radical is the only species lik ely to drive the dealkylation chemistry for this model substrate. The final chapter seeks to better unde rstand the relationship between the benzaldehyde imino-oxy acetic acid and N -benzoylglycine substrates by using a novel approach to structure-activit y relationships. Using high level docking algorithms, the binding orientation for a series of molecules that structurally linked Nbenzoylglycine to benzaldehyde imino-oxy acetic acid through combinations of oxygen presence/absence (example O -acetyl hydroxamic acid and imine-cont aining derivatives). These poses 37

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CHAPTER 1: Introduction were further analyzed using Natural Bond An alysis (NBO) to look at the lone pair contribution of each structure to determine th e role of hyperconjugative stabilization. The relationship of activity to the electron ic information provided through NBO analysis suggest that benzaldehyde imino-oxy acetic acid is in fact electronically very similar to N -benzoylglycine. This structure-activity study also contained many imino-oxy acetic derivatives which were shown to have simila r inhibition to Michaelis constant values, suggesting that only one bindi ng orientation exists for these compounds and that the (VMAX/KM)apparent values for these compounds ar e equal to or better than N -benzoylglycine analogues. These kinetic and theoretical results make the imino-oxy acetic acid an interesting moiety for dr ug design targeting PAM. 38

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CHAPTER 1: Introduction References (1) Glembotski, C. C. (1986) The characterization of the ascorbic acid-mediated alpha-amidation of alpha-melanotropin in cultured intermediate pituitary lobe cells. Endocrinology 118 1461-8. (2) Habermann, E., and Jentsch, J. (1967) Se quential analysis of melittin from tryptic and peptic fragments. Hoppe-Seyler's Zeitschrift fuer Physiologische Chemie 348, 37-50 (3) Bradbury, A. F., Finnie, M. D., and Smyth, D. G. (1982) Mechanism of Cterminal amide formation by pituitary enzymes. Nature 298, 686-8. (4) Bolkenius, F. N., and Ganzhorn, A. J. (1998) Peptidylglycine alpha-amidating mono-oxygenase: neuropeptide amidation as a target for drug design. Gen Pharmacol 31, 655-9. (5) Braas, K. M., Harakall, S. A., Ouafi k, L., Eipper, B. A., and May, V. (1992) Expression of peptidylglycine alpha -amidating monooxygenase: an in situ hybridization and immunocytochemical study. Endocrinology 130, 2778-88. (6) Prigge, S. T., Mains, R. E., Eipper, B. A., and Amzel, L. M. (2000) New insights into copper monooxygenases and peptide amidation: structure, mechanism and function. Cell Mol Life Sci 57, 1236-59. (7) Merkler, D. J., Chew, G. H., Gee, A. J., Merkler, K. A., Sorondo, J. P., and Johnson, M. E. (2004) Oleic acid derived metabolites in mouse neuroblastoma N18TG2 cells. Biochemistry 43, 12667-74. (8) Merkler, K. A., Baumgart, L. E., De Blassio, J. L., Glufke U., King, L., 3rd, Ritenour-Rodgers, K., Vederas, J. C., Wilc ox, B. J., and Merkler, D. J. (1999) A pathway for the biosynthesis of fatty acid amides. Adv Exp Med Biol 469, 519-25. (9) Wilcox, B. J., Ritenour-Rodgers, K. J., A sser, A. S., Baumgart, L. E., Baumgart, M. A., Boger, D. L., DeBlassio, J. L., deLong, M. A., Glufke, U., Henz, M. E., King, L., 3rd, Merkler, K. A., Patterson, J. E ., Robleski, J. J., Vederas, J. C., and Merkler, D. J. (1999) N-acylglycine amid ation: implications for the biosynthesis of fatty acid primary amides. Biochemistry 38, 3235-45. (10) Merkler, D. J., Merkle r, K. A., Stern, W., and Flem ing, F. F. (1996) Fatty acid amide biosynthesis: a possible new role for peptidylglycine alpha-amidating enzyme and acyl-coenzyme A: glycine N-acyltransferase. Arch Biochem Biophys 330, 430-4. (11) King, L., 3rd, Barnes, S., Glufke, U., Henz, M. E., Kirk, M., Merkler, K. A., Vederas, J. C., Wilcox, B. J., and Merkle r, D. J. (2000) The enzymatic formation of novel bile acid primary amides. Arch Biochem Biophys 374 107-17. (12) Chaturvedi, S., Driscoll, W. J., Ellio t, B. M., Faraday, M. M., Grunberg, N. E., and Mueller, G. P. (2006) In vivo evidence that N-oleoylglycine acts independently of its conversion to oleamide. Prostaglandins Other Lipid Mediat 81, 136-49. (13) Ritenour-Rodgers, K. J., Driscoll, W. J ., Merkler, K. A., Merkler, D. J., and Mueller, G. P. (2000) Induction of peptidylglycine alpha-amidating monooxygenase in N(18)TG(2) cells: a model for studying oleamide biosynthesis. Biochem Biophys Res Commun 267, 521-6. 39

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CHAPTER 1: Introduction (14) Walsh, C. T. (2006) Posttranslational Modifications of Proteins: Expanding Nature's Inventory, Roberts and Company Publishers, Englewood, Colorado. (15) Rush, R. A., and Geffen, L. B. (1980) Dopamine beta-hydroxylase in health and disease. Crit Rev Clin Lab Sci 12, 241-77. (16) Klinman, J. P. (1996) Mechanisms Whereby Mononuclear Copper Proteins Functionalize Organic Substrates. Chem Rev 96, 2541-2562. (17) Klinman, J. P. (2006) The co pper-enzyme family of dopamine betamonooxygenase and peptidylglycine alpha-hydroxylating monooxygenase: resolving the chemical pathwa y for substrate hydroxylation. J Biol Chem 281, 3013-6. (18) Stewart, L. C., and Klinman, J. P. (1988) Dopamine beta-hydroxylase of adrenal chromaffin granules: structure and function. Annu Rev Biochem 57, 551-92. (19) Carmicheal, S. W., and Winkler, H. (1985) The adrenal chromaffin cell. Scientific American 253, 40-49. (20) Nakamura, S. (1990) Senile dementia and presenile dementia. Tohoku J Exp Med 161 Suppl 49-60. (21) Nagatsu, T., and Sawada, M. (2007) Biochemistry of postmortem brains in Parkinson's disease: historical overview and future prospects. J Neural Transm Suppl 113-20. (22) Honecker, H., Fahndrich, E., Coper, H., and Helmchen, H. (1981) Serum DBH and platelet MAO in patients with depressive disorders. Pharmacopsychiatria 14, 10-4. (23) Southan, C., and Kruse, L. I. (1989) Sequence similarity between dopamine betahydroxylase and peptide alpha-amidatin g enzyme: evidence for a conserved catalytic domain. FEBS Lett 255, 116-20. (24) Kulathila, R., Consalvo, A. P., Fitzpa trick, P. F., Freeman, J. C., Snyder, L. M., Villafranca, J. J., and Merkler, D. J. (1994) Bifunctional peptidylglcine alphaamidating enzyme requires two copper atoms for maximum activity. Arch Biochem Biophys 311, 191-5. (25) Klinman, J. P., Krueger, M., Brenner, M., and Edmondson, D. E. (1984) Evidence for two copper atoms/subunit in dopamine beta-monooxygenase catalysis. J Biol Chem 259, 3399-402. (26) Ash, D. E., Papadopoulos, N. J., Colo mbo, G., and Villafranca, J. J. (1984) Kinetic and spectroscopic stud ies of the interaction of copper with dopamine betahydroxylase. J Biol Chem 259, 3395-8. (27) Prigge, S. T., Eipper, B. A., Main s, R. E., and Amzel, L. M. (2004) Dioxygen binds end-on to mononuclear copper in a precatalytic enzyme complex. Science 304, 864-7. (28) Prigge, S. T., Kolhekar, A. S., Eipper B. A., Mains, R. E., and Amzel, L. M. (1999) Substrate-mediated electron tr ansfer in peptidylglycine alphahydroxylating monooxygenase. Nat Struct Biol 6, 976-83. (29) Blackburn, N. J., Hasnain, S. S., Pettin gill, T. M., and Strange, R. W. (1991) Copper K-extended x-ray absorption fine structure studies of oxidized and reduced dopamine beta-hydroxylase. Confirma tion of a sulfur ligand to copper(I) in the reduced enzyme. J Biol Chem 266, 23120-7. 40

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CHAPTER 1: Introduction (30) Blumberg, W. E., Desai, P. R., Powers, L., Freedman, J. H., and Villafranca, J. J. (1989) X-ray absorption spectroscopic st udy of the active copper sites in dopamine beta-hydroxylase. J Biol Chem 264, 6029-32. (31) Jaron, S., and Blackburn, N. J. (2001) Characterization of a half-apo derivative of peptidylglycine monooxygenase. Insight into the reactivity of each active site copper. Biochemistry 40, 6867-75. (32) Kulathila, R., Merkler, K. A., and Me rkler, D. J. (1999) Enzymatic formation of C-terminal amides. Nat Prod Rep 16, 145-54. (33) Rhames, F. C., Murthy, N. N., Kar lin, K. D., and Blackburn, N. J. (2001) Isocyanide binding to the coppe r(I) centers of the catalytic core of peptidylglycine monooxygenase (PHMcc). J Biol Inorg Chem 6, 567-77. (34) Francisco, W. A., Wille, G., Smith, A. J., Merkler, D. J., and Klinman, J. P. (2004) Investigation of the pathway fo r inter-copper electron transfer in peptidylglycine alpha-amidating monooxygenase. J Am Chem Soc 126, 13168-9. (35) Francisco, W. A., Merkler, D. J., Blackburn, N. J., and Klinman, J. P. (1998) Kinetic mechanism and intrinsic isotope effects for the peptidylglycine alphaamidating enzyme reaction. Biochemistry 37, 8244-52. (36) Owen, T. C., and Merkler, D. J. (2004) A new proposal for the mechanism of glycine hydroxylation as catalyzed by peptidylglycine alpha-hydroxylating monooxygenase (PHM). Med Hypotheses 62, 392-400. (37) Eipper, B. A., Perkins, S. N., Huste n, E. J., Johnson, R. C., Keutmann, H. T., and Mains, R. E. (1991) Peptidyl-alpha -hydroxyglycine alpha-amidating lyase. Purification, characterization, and expression. J Biol Chem 266, 7827-33. (38) De, M., Bell, J., Blackbur n, N. J., Mains, R. E., and Ei pper, B. A. (2006) Role for an essential tyrosine in peptide amidation. J Biol Chem 281, 20873-82. (39) Katopodis, A. G., Ping, D., and May, S. W. (1990) A novel enzyme from bovine neurointermediate pituitary catalyzes dealkylation of alpha-hydroxyglycine derivatives, thereby functioning seque ntially with peptidylglycine alphaamidating monooxygenase in peptide amidation. Biochemistry 29, 6115-20. (40) Bell, J., Ash, D. E., Sn yder, L. M., Kulathila, R., Bl ackburn, N. J., and Merkler, D. J. (1997) Structural and functional i nvestigations on the role of zinc in bifunctional rat peptidylglyc ine alpha-amidating enzyme. Biochemistry 36, 16239-46. (41) Moore, A. B., and May, S. W. (1999) Kinetic and inhibition studies on substrate channelling in the bifunctional enzy me catalysing C-terminal amidation. Biochem J 341 ( Pt 1) 33-40. (42) Smith, S. J., Casellato, A., Hadler, K. S ., Mitic, N., Riley, M. J., Bortoluzzi, A. J., Szpoganicz, B., Schenk, G., Neves, A., and Gahan, L. R. (2007) The reaction mechanism of the Ga(III)Zn(II) deriva tive of uteroferrin and corresponding biomimetics. J Biol Inorg Chem 12, 1207-20. (43) Neves, A., Lanznaster, M., Bortoluzzi, A. J., Peralta, R. A., Casellato, A., Castellano, E. E., Herra ld, P., Riley, M. J., and Schenk, G. (2007) An unprecedented Fe(III)(mu-OH)Zn(II) complex that mimics the structural and functional properties of purple acid phosphatases. J Am Chem Soc 129, 7486-7. 41

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CHAPTER 1: Introduction (44) Funhoff, E. G., Bollen, M., and Averill, B. A. (2005) The Fe(III)Zn(II) form of recombinant human purple acid phosphata se is not activated by proteolysis. J Inorg Biochem 99, 521-9. (45) Lanznaster, M., Neves, A., Bortoluzzi, A. J., Szpoganicz, B., and Schwingel, E. (2002) New Fe(III)Zn(II) complex containi ng a single terminal Fe-O(phenolate) bond as a structural and functional model for the active site of red kidney bean purple acid phosphatase. Inorg Chem 41, 5641-3. (46) Katopodis, A. G., and May, S. W. (1990) Novel substrates and inhibitors of peptidylglycine alpha-amidating monooxygenase. Biochemistry 29, 4541-8. (47) May, S. W., Mueller, P. W., Padgette, S. R., Herman, H. H., and Phillips, R. S. (1983) Dopamine-B-hydroxylase: suicide inhibition by the novel olefinic substrate, 1-phenyl-1-aminomethylethene. Biochem Biophys Res Commun 110, 161-8. (48) Padgette, S. R., Wimalasena, K., Herman, H. H., Sirimanne, S. R., and May, S. W. (1985) Olefin oxygenation and N-dealkylation by dopamine betamonooxygenase: catalysis and mechanism-based inhibition. Biochemistry 24, 5826-39. (49) Sirimanne, S. R., and May, S. W. (1995) Interaction of non-conjugated olefinic substrate analogues with dopamine beta-monooxygenase: catalysis and mechanism-based inhibition. Biochem J 306 ( Pt 1) 77-85. (50) May, S. W., Herman, H. H., Roberts, S. F., and Ciccarello, M. C. (1987) Ascorbate depletion as a consequence of product recycling during dopamine betamonooxygenase catalyzed selenoxidation. Biochemistry 26, 1626-33. (51) May, S. W., Phillips, R. S., Mueller, P. W., and He rman, H. H. (1981) Dopamine beta-hydroxylase. Demonstration of enzymatic ketonization of the product enantiomer S-octopamine. J Biol Chem 256, 2258-61. (52) May, S. W., Phillips, R. S., Herma n, H. H., and Mueller, P. W. (1982) Bioactivation of Catha edulis alkaloids: enzymatic ketonization of norpseudoephedrine. Biochem Biophys Res Commun 104, 38-44. (53) Klinman, J. P., and Krueger, M. (1982) Dopamine beta-hydroxylase: activity and inhibition in the presence of beta-substituted phenethylamines. Biochemistry 21 67-75. (54) Mangold, J. B., and Klinman, J. P. (1984) Mechanism-based inactivation of dopamine beta-monooxygenase by beta-chlorophenethylamine. J Biol Chem 259, 7772-9. (55) Bossard, M. J., and Klinman, J. P. (1986) Mechanism-based inhibition of dopamine beta-monooxygenase by aldehydes and amides. J Biol Chem 261, 16421-7. (56) Sirimanne, S. R., and May, S. W. (1988) Facile stereo selective allylic hydroxylation by dopamine beta-monooxygenase. J Amer Chem Soc 110 75607561. (57) Katopodis, A. G., and May, S. W. (1988) A new facile trinitrophenylated substrate for peptide alpha-amidation and its use to characterize PAM activity in chromaffin granules. Biochem Biophys Res Commun 151, 499-505. (58) Silverman, R. B. (2002) The organic chemistry of enzyme-catalyzed reactions Academic Press. 42

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CHAPTER 1: Introduction (59) Hillas, P. J., and Fitzpatrick, P. F. (1996) A mechan ism for hydroxylation by tyrosine hydroxylase based on partitioning of substituted phenylalanines. Biochemistry 35, 6969-75. (60) Frantom, P. A., Seravalli, J., Ragsda le, S. W., and Fitzpatrick, P. F. (2006) Reduction and oxidation of the active site iron in tyrosine hydroxylase: kinetics and specificity. Biochemistry 45, 2372-9. (61) Ellis, H. R., McCusker, K. P., and Fitzpatrick, P. F. (2002) Use of a tyrosine hydroxylase mutant enzyme with reduced metal affinity allows detection of activity with cobalt in place of iron. Arch Biochem Biophys 408, 305-7. (62) Eser, B. E., Barr, E. W., Frantom, P. A., Saleh, L., Bollinger, J. M., Jr., Krebs, C., and Fitzpatrick, P. F. (2007) Direct Spectroscopic Evidence for a High-Spin Fe(IV) Intermediate in Tyrosine Hydroxylase. J Am Chem Soc 129, 11334-5. (63) Murray, L. J., and Lippard, S. J. (2007) Substrate tr afficking and dioxygen activation in bacterial multicomponent monooxygenases. Acc Chem Res 40, 46674. (64) Hakemian, A. S., and Rosenzweig, A. C. (2007) The biochemistry of methane oxidation. Annu Rev Biochem 76, 223-41. (65) Baik, M. H., Newcomb, M., Friesner, R. A., and Lippard, S. J. (2003) Mechanistic studies on the hydroxyl ation of methane by methane monooxygenase. Chem Rev 103, 2385-419. (66) Kovaleva, E. G., Neibergall, M. B., Ch akrabarty, S., and Lipscomb, J. D. (2007) Finding intermediates in the O2 activ ation pathways of non-heme iron oxygenases. Acc Chem Res 40, 475-83. (67) Rinaldo, D., Philipp, D. M., Lippar d, S. J., and Friesner, R. A. (2007) Intermediates in dioxygen activatio n by methane monooxygenase: a QM/MM study. J Am Chem Soc 129, 3135-47. (68) Bollinger, J. M., Jr., and Krebs, C. (2006) Stalking intermediates in oxygen activation by iron enzymes: motivation and method. J Inorg Biochem 100, 586605. (69) Decker, H., Schweikardt, T., and Tuczek, F. (2006) The first crystal structure of tyrosinase: all questions answered? Angew Chem Int Ed Engl 45, 4546-50. (70) Solomon, E. I., Sundaram, U. M., and Machonkin, T. E. (1996) Multicopper Oxidases and Oxygenases. Chem Rev 96, 2563-2606. (71) Land, E. J., Ramsden, C. A., and Riley, P. A. (2003) Tyrosinase autoactivation and the chemistry of ortho-quinone amines. Acc Chem Res 36, 300-8. (72) Matoba, Y., Kumagai, T., Yamamoto, A., Yoshitsu, H., and Sugiyama, M. (2006) Crystallographic evidence that the dinuclear copper center of tyrosinase is flexible during catalysis. J Biol Chem 281, 8981-90. (73) Decker, A., and Solomon, E. I. (200 5) Dioxygen activation by copper, heme and non-heme iron enzymes: comparison of electronic structures and reactivities. Curr Opin Chem Biol 9, 152-63. (74) Lehnert, N., George, S. D., and So lomon, E. I. (2001) Recent advances in bioinorganic spectroscopy. Curr Opin Chem Biol 5, 176-87. (75) Rompel, A., Fischer, H., Meiwes, D ., Buldt-Karentzopoulos, K., Dillinger, R., Tuczek, F., Witzel, H., and Krebs, B. (1999) Purification and spectroscopic studies on catechol oxidases from Lyc opus europaeus and Populus nigra: 43

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CHAPTER 1: Introduction evidence for a dinuclear copper center of t ype 3 and spectroscopic similarities to tyrosinase and hemocyanin. J Biol Inorg Chem 4, 56-63. (76) Hirota, S., Kawahara, T., Lonardi, E., de Waal, E., Funasaki, N., and Canters, G. W. (2005) Oxygen binding to tyrosinase from streptom yces antibioticus studied by laser flash photolysis. J Am Chem Soc 127, 17966-7. (77) Xin, X., Mains, R. E., and Eipper, B. A. (2004) Monooxygenase X, a member of the copper-dependent monooxygenase family localized to the endoplasmic reticulum. J Biol Chem 279, 48159-67. (78) Hess, C. R., McGuirl, M. M., and Klin man, J. P. (2008) Mechanism of the Insect Enzyme, Tyramine {beta}-Monooxygena se, Reveals Differences from the Mammalian Enzyme, Dopamine {beta}-Monooxygenase. J Biol Chem 283, 30429. (79) Knecht, A. K., and Bronner-Fraser, M. (2001) DBHR, a gene with homology to dopamine beta-hydroxylase, is expressed in the neural cres t throughout early development. Dev Biol 234 365-75. (80) Prohaska, J. R., and Gybina, A. A. (2004) Intracellular copper transport in mammals. J Nutr 134, 1003-6. (81) El Meskini, R., Culotta, V. C., Mains, R. E., and Eipper, B. A. (2003) Supplying copper to the cuproenzyme peptidyl glycine alpha-amidating monooxygenase. J Biol Chem 278, 12278-84. (82) Steveson, T. C., Ciccotosto, G. D., Ma, X. M., Mueller, G. P., Mains, R. E., and Eipper, B. A. (2003) Menkes protei n contributes to the function of peptidylglycine alpha-amidating monooxygenase. Endocrinology 144, 188-200. (83) Merkler, D. J., Asser, A. S., Baumgart, L. E., Carpenter, S. E., Chew, G. H., Consalvo, A. P., DeBlassio, J. L., Galloway, L. C., Lowe, A. B., Lowe, E. W. J., Lawrence King III, L., Kendig, R. D., Kline, P. C., Kulathila, R., Robert Malka, R., Merkler, K. A., McIntyre, N. R., Wilcox, B. J., and Owen, T. C. Hippurate, Substituted Hippuates, and Hippurate Analog s as Substrates and Inhibitors of Peptidylglycine -Hydroxylating Monooxygenase (PHM). in preparation. (84) Tamburini, P. P., Young, S. D., Jones, B. N., Palmesino, R. A., and Consalvo, A. P. (1990) Peptide substrate specificity of the alpha-amidating enzyme isolated from rat medullary thyroid CA-77 cells. Int J Pept Protein Res 35, 153-6. (85) Bradbury, A. F., and Smyt h, D. G. (1983) Substrate-sp ecificity of an amidating enzyme in porcine pituitary. Biochem Biophys Res Commun 117, 372-377. (86) Jaron, S., and Blackburn, N. J. (1999) Does superoxide channel between the copper centers in peptidylglycine m onooxygenase? A new mechanism based on carbon monoxide reactivity. Biochemistry 38, 15086-96. (87) Prigge, S. T., Kolhekar, A. S., Eipper B. A., Mains, R. E., and Amzel, L. M. (1997) Amidation of bioactive peptides: the structure of peptidylglycine alphahydroxylating monooxygenase. Science 278, 1300-5. (88) Blackburn, N. J., Rhames, F. C., Ralle, M., and Jaron, S. (2000) Major changes in copper coordination accompany reducti on of peptidylglycine monooxygenase: implications for electron transfer and the catalytic mechanism. J Biol Inorg Chem 5, 341-53. 44

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CHAPTER 1: Introduction (89) Glembotski, C. C. (1987) The role of ascorbic acid in the biosynthesis of the neuroendocrine peptides alpha-MSH and TRH. Ann N Y Acad Sci 498, 54-62. (90) Brenner, M. C., and Klinman, J. P. (1989) Correlation of copper valency with product formation in single turnovers of dopamine beta-monooxygenase. Biochemistry 28, 4664-70. (91) Klinman, J. P., and Brenner, M. (19 88) Role of copper an d catalytic mechanism in the copper monooxygenase, dopamine beta-hydroxylase (D beta H). Prog Clin Biol Res 274 227-48. (92) Brenner, M. C., Murray, C. J., and Klinman, J. P. (1989) Rapid freezeand chemical-quench studies of dopamine beta-monooxygenase: comparison of presteady-state and steady-state parameters. Biochemistry 28, 4656-64. (93) Klinman, J. P. (1978) Kineti c isotope effects in enzymology. Adv Enzymol Relat Areas Mol Biol 46, 415-94. (94) Berti, P. J. (1999) Determining transi tion states from kinetic isotope effects. Methods Enzymol 308, 355-97. (95) Harris, D. C. a. B., M.D. (1978) Symmetry and Spectroscopy: An Introduction to Vibrational and Electronic Spectroscopy Oxford university Press, Inc., New York. (96) Huskey, W. P. (1991) Origins and Interpretations of Heavy-Atom Isotope Effects CRC Press, Inc., Boca Roton. (97) Suhnel, J. a. S., R.L (1991) Theoretical Basis for Primary and Secondary Hydrogen Isotope Effects, ed., CRC Press, Inc., Boca Raton. (98) Schramm, V. L. (2005) Enzymatic tran sition states and transition state analogues. Curr Opin Struct Biol 15 604-13. (99) Schramm, V. L. (2005) Enzymatic transition states: thermodynamics, dynamics and analogue design. Arch Biochem Biophys 433 13-26. (100) Schramm, V. L. (2003) Enzymatic tr ansition state poise and transition state analogues. Acc Chem Res 36, 588-96. (101) Schramm, V. L., and Shi, W. ( 2001) Atomic motion in enzymatic reaction coordinates. Curr Opin Struct Biol 11 657-65. (102) Schramm, V. L. (2001) Transition state variation in enzymatic reactions. Curr Opin Chem Biol 5, 556-63. (103) Schramm, V. L. (1998) Enzymatic transition states and transition state analog design. Annu Rev Biochem 67, 693-720. (104) Francisco, W. A., Knapp, M. J., Bl ackburn, N. J., and Klinman, J. P. (2002) Hydrogen tunneling in peptidylglyc ine alpha-hydroxylating monooxygenase. J Am Chem Soc 124, 8194-5. (105) Knapp, M. J., and Klinman, J. P. (2002) Environmentally coupled hydrogen tunneling. Linking catalysis to dynamics. Eur J Biochem 269, 3113-21. (106) Knapp, M. J., Rickert, K., and Klin man, J. P. (2002) Temperature-dependent isotope effects in soybean lipoxygenase-1 : correlating hydrogen tunneling with protein dynamics. J Am Chem Soc 124, 3865-74. (107) Chen, P., and Solomon, E. I. (2004) O2 activation by binuclear Cu sites: noncoupled versus exchange coupled reaction mechanisms. Proc Natl Acad Sci U S A 101, 13105-10. 45

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CHAPTER 1: Introduction (108) Chen, P., and Solomon, E. I. (2004) Oxygen activation by the noncoupled binuclear copper site in peptidylglycine alpha-hydroxylat ing monooxygenase. Reaction mechanism and role of the nonc oupled nature of the active site. J Am Chem Soc 126, 4991-5000. (109) Crespo, A., Marti, M. A ., Roitberg, A. E., Amzel, L. M. and Estrin, D. A. (2006) The catalytic mechanism of peptidylglycine alpha-hydroxylating monooxygenase investigated by computer simulation. J Am Chem Soc 128, 12817-28. (110) Yoshizawa, K., Kihara, N., Kamach i, T., and Shiota, Y. (2006) Catalytic mechanism of dopamine beta-m onooxygenase mediated by Cu(III)-oxo. Inorg Chem 45, 3034-41. (111) Kamachi, T., Kihara, N., Shiota, Y. and Yoshizawa, K. (2005) Computational exploration of the catalytic mechanism of dopamine beta-monooxygenase: modeling of its mononuclear copper active sites. Inorg Chem 44, 4226-36. (112) Bollinger, J. M., Jr., and Krebs, C. (2007) Enzymatic C-H activation by metalsuperoxo intermediates. Curr Opin Chem Biol 11, 151-8. (113) Roth, J. P. (2007) Advances in studying bioinorganic reaction mechanisms: isotopic probes of activated oxygen intermediates in metalloenzymes. Curr Opin Chem Biol (114) Francisco, W. A., Blackburn, N. J ., and Klinman, J. P. (2003) Oxygen and hydrogen isotope effects in an active site tyrosine to phenyl alanine mutant of peptidylglycine alpha-hydroxylating monooxyg enase: mechanistic implications. Biochemistry 42, 1813-9. (115) Miller, S. M., and Klinman, J. P. (1983) Magnitude of intrinsic isotope effects in the dopamine beta-monooxygenase reaction. Biochemistry 22 3091-6. (116) Tian, G., Berry, J. A., and Klinma n, J. P. (1994) Oxygen-18 kinetic isotope effects in the dopamine beta-monooxygena se reaction: evidence for a new chemical mechanism in non-heme metallomonooxygenases. Biochemistry 33, 226-34. (117) Evans, J. P., Ahn, K., and Klinman, J. P. (2003) Evidence that dioxygen and substrate activation are tightly c oupled in dopamine beta-monooxygenase. Implications for the reactive oxygen species. J Biol Chem 278, 49691-8. (118) Fitzpatrick, P. F., Flory, D. R., Jr., a nd Villafranca, J. J. (1985) 3-Phenylpropenes as mechanism-based inhibitors of dopa mine beta-hydroxylase: evidence for a radical mechanism. Biochemistry 24 2108-14. (119) Miller, S. M., and Klin man, J. P. (1985) Secondary isotope effects and structurereactivity correlations in the dopamine beta-monooxygenase reaction: evidence for a chemical mechanism. Biochemistry 24, 2114-27. (120) Fitzpatrick, P. F., and Villafranca, J. J. (1987) Mechanism-based inhibitors of dopamine beta-hydroxylase. Arch Biochem Biophys 257, 231-50. (121) Wimalasena, K., and May, S. W. (1989) Dopamine beta-monooxygenase catalyzed aromatization of 1-(2-aminoeth yl)-1,4-cyclohexadiene redirection of specificity and evidence for a hydr ogen-atom transfer mechanism. J Am Chem Soc 111, 2729-2731. (122) Wimalasena, K., and May, S. W. (1989) Dopamine -Monooxygenase Catalyzed Aromatization of 1-(2-Aminoethyl)-1,4 -Cyclohexadiene: Redirection of 46

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CHAPTER 1: Introduction Specificity and Evidence for a Sing le Electron Transfer Mechanism. J. Am. Chem. Soc. 2729. (123) Chen, P., Root, D. E., Campochiaro, C ., Fujisawa, K., and Solomon, E. I. (2003) Spectroscopic and electronic structure st udies of the diama gnetic side-on CuIIsuperoxo complex Cu(O2)[HB(3-R-5-iPrpz) 3]: antiferromagnetic coupling versus covalent delocalization. J Am Chem Soc 125, 466-74. (124) Chen, P., and Solomon, E. I. (2002) Fr ontier molecular orbita l analysis of Cu(n)O(2) reactivity. J Inorg Biochem 88, 368-74. (125) Maiti, D., Fry, H. C., Woertink, J. S., Vance, M. A., Solomon, E. I., and Karlin, K. D. (2007) A 1:1 copper-dioxyge n adduct is an end-on bound superoxo copper(II) complex which undergoes oxygenation reactions with phenols. J Am Chem Soc 129, 264-5. 47

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CHAPTER 2: Substrate Pre-Organization in PHM Introduction In comparison to peptide hormones, cu rrent biosynthetic pathways for primary fatty acid amides are poorly understood ( 1). The Merkler group has postulated the formation of both conjugated/non-conjugated glycine-extended fatty acid and bile acid amides from their CoA thioester derivatives by acyl-CoA:glycine N -acyltransferase (ACGNAT) (E.C. 2.3.1.13) ( 2-5 ) and bile acid coenzyme A:amino acid N -acyltransferase (BAAT) (E.C. 2.3.1.65) (6-8 ). Each of these enzyme s catalyzes the sequential thioesterase and acyl transferase reactions required for glycine coupling. This glycineextended product is then used as a substrate for peptidylglycine -amidating monooxygenase (PAM) rendering the corresponding amide and glyoxylate products. The most compelling evidence for the biosynthetic pathway of fatty acid derivatives utilizing PAM was observed with the sleep regulating, fatty acid hormone, Noleamide (Fig. 2.1.) ( 9-11). CH3(CH2)7O NH2 (CH2)7 Figure 2.1. Oleamide ( cis -9-octadecenamide) The addition of exogenous, radio-labeled oleic acid displayed a PAM dependent conversion to Noleamide which suggested that Noleoylglycine was an intermediate in a novel biosynthetic and regulatory pathway for this fatty acid amide( 5). 47

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CHAPTER 2: Substrate Pre-Organization in PHM Gl y cineCoA-SH 2H 2e, O2H2O PHM PAL 2CuIIZnII, FeIIIPathway A: ACNAT Pathway B: BAAT A. Fatty Nacyl-CoA B. Bile Nacyl-CoA A. Fatty Nacylglycine B. Bile Nacylglycine A. Fatty Nacylamide B. Bile Nacylamide+ glyoxylat e Scheme 2.1. Postulated pathway for the reduction of both fatty and bile Nacyl-CoA to their corresponding amide and glyoxylate products. Although not displayed, an ATP-dependent step is hypothesized to catalyze the formation of the acyl-CoA thioester used with ACNAT and BAAT. As Nglycine-extended derivatives of fatty and bile acids have been shown to undergo oxidative cleavage via PAM (schem e 2.1) ( 2-4, 6, 7, 12), this pathway has been further hypothesized to include fatty acid amide hydrolase (FAAH)( 13). FAAH is an amidase signature (AS) enzyme( 14, 15), and its role for following PAM in lipid amide signaling regulation, appears plausible( 3, 4, 6). Critical for complete understa nding of this pathway is the kinetic mechanism for both dopamine -monooxygenase (D M) and peptidylglycine -hydroxylating monooxygenase (PHM), the monooxygenase domain of PAM. Both enzymes have been studied extensively, though many hypotheses have been difficult to fully elucidate due to the extreme complexity of each systems( 16-18). Excluding substr ate specificity of D M and PHM, all other salient features of each reaction in cluding copper-mediated oxygen activation, C -H transfer and hydroxylation mechan isms, have been experimentally observed to be super-imposable (equation 2.1.)(17, 18 ). RH 2e-, 2H+, O22CuIIH2O D M or PHM RO H Scheme 2.2. Representative reaction for th e hydrogen transfer/s ubstrate oxidation reaction for D M (R=benzyl donors of phenyl ethylamines) and PHM (R= -glycyl donors of glycine-extended s ubstrates), respectively. 48

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CHAPTER 2: Substrate Pre-Organization in PHM Semi-classical formalisms such as the transition-state theory (TST) and variational transition state th eory (VTST) are unable to fully explain the wealth of mechanistic data for both PHM and D M. For example, the large intrinsic and secondary kinetic isotope effects associated with C -H cleavage (chemical step) are beyond semiclassical values and independent of temperature( 19, 20). As a result, PHM and D M are best treated with a modified Marcus-model to understand th e quantum mechanical nature of substrate and protein within the activation of each system( 20-22). Marcus theory governs the environmental factors employed to develop the transi tion configuration required for hydrogen transfer. The rate limiting step with in hydrogen transfer is the probability of attaining the transition configuration for this coordinate( 22, 23). Specifically, the potential energy surface of the hydrogen transfer coordinate is controlled by the environmental surface (prote in and solvent). Within Ma rcus theory, the de Broglie wavelength of the hydrogen donor, defines not only the wavefunction of the transferred particle but as we will see, has a great deal of influence modulating the degenerate wave function overlap necessary to reach the transition configuration (figure 2.3)( 24). As the degenerate overlap between donor and acceptor is highly sensitive to distance, isotopologues of hydrogen yield decr easing de Broglie wavelengths ( ) proportional to mass ( = 0.50 (H), 0.31 (D), and 0.25 (T))(25). Large primary kinetic isotope effects for these reactions signify transfer c oordinates which are similar in magnitude to the hydrogen wavelength, respectively ( 26). 49

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CHAPTER 2: Substrate Pre-Organization in PHM Figure 2.2 The two parabolas represent the reactant (R) and product (P) wells in the Marcus-model for electron transfer. The G term represents the barrier for the electr on transfer process. This activation term is dependent on both reaction driving force ( G) and reorganization energy ( ), respectively. As displayed in figure 2.2, th e Marcus model for electron tr ansfer between two parabolic states within a reaction coordinate are determined by three interconnected thermodynamic parameters. GG GN`a2/ 4 Equation 2.1. Marcus equation of the thermodynamics governing electron transfer. k exp@ GGRTfffffffffffffffffffff fg exp@ GObc24 RTfffffffffffffffffffffffffffffffffff Equation 2.2. Marcus equation for the probab ility of electron transfer. The first, reorga nizatio n energy ( ), is the energy required to transfer the hydrogen particle from the bottom of the reactant ener gy profile state (R) to the energy profile of the acceptor state (P) (figure 2.2.). Once this activation energy requirement ( G) is met, the reactant and product pa rabolas will be of equal free ener gy allowing transfer between the barriers to occur. The inter-relationship of both G and is described in equation 2.1 under electron transfer formalisms. Their de pendence on the total free energy change for the process ( G) has a net free energy equal to zero when the activation free energy 50

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CHAPTER 2: Substrate Pre-Organization in PHM equals /4, respectively(27). The probability of electron tunneling is treated as a rate expression in equation 2.3. For hydrogen atom transfer, the Marcus m odel must be modified from equation 2.2 using the formalisms derive d by Kuznetsov and Ulstrup( 28). Equation 2.4 describes the tunneling probability re quired for hydrogen tr ansfer under the modified Marcus model used by Knapp et al. ( 23). ktunXXw1 2fffffffffVelL L L M M M243 RT -2fffffffffffffffffffff v u u t wwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwexp@ Go Evibbc24 RTfffffffffffffffffffffffffffffffffffffffffffffffffffffffffffDEx F A C A term`av,w Equation 2.3. Tunneling probability for hydrogen transfer using the modified Marcus equation as described by Knapp et al. ( 23). This equation contains the Marcus terms (shown in square parentheses) as described in equations 1 and 2 with the Franck-Condon term. The tunneling probability is determined from the total sum of electronic, vibrational and nuclear r eactant and product states al ong a reaction coordinate. Please note that and w are the vibrational product of both reactan t and product, respectively. These terms have been normalized accordin g to their thermal position (P ). | Vel| 2 and Evib describe the reactant and product electronic overlap and vibrational energy components. For the quantum mechanical tunneli ng of hydrogen, the Franck-Condon term is paramount to determining tunneling probabi lity for hydrogen transfer and understanding the kinetic isotope effects therei n. This term states that lig ht particle transitions occur only near heavy nuclear confi gurations in which the light particle energies match( 28). This principle is based on se parating out the slow coordina tes involved with protein and solvent dynamics from the fast quantum mechanical coordi nates involved in hydrogen transfer. This degenerate wave function overlap between donor and acceptor only occurs at the transition configuration (figure 2.3). This event result s in the requisite energy to reach the transition state (G, free energy of activation, figure 2.2) and promote a through barrier or tunneling process. For the transfer of hydrogen or hydrogenic 51

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CHAPTER 2: Substrate Pre-Organization in PHM isotopes, the integrated Franck Condon term (equation 2.4b) allows the mass, temperature factors to be separated and eval uated in terms of hydrogen tunneling factors. Overall, the tunneling probability (equation 2.3) is governed by an isotope independent and temperature dependent Marcus term coupled to the Franck Condon term which is mass dependent (temperature independent in 4a and dependent in its integrated form) while also reflecting the distance between donor and acceptor in the transfer coordinate. F A C A term0,0 exp @ mHH r2b2 -c f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f (a) F A C A term0,0 Z 0 r0exp@ m1H r22 -ffffffffffffffffffffffffffffffffffffffffexp@HX22 RTffffffffffffffffffffffffffffffffdx (b) Equation 2.4. (a) Franck Condon terms representing the wave function overlap between donor and acceptor as a function of mass (mH), frequency ( H), and transfer distance from donor ( r). represents the value of Plancks constant over h/ 2 (1.05457 x 10-34 a J s). (b) Integration of the Franck Condon term in equation 4a. This operation is used to express the tunneling quation 4b) as a function of distance between donor and acceptor. In this equation, where rx, mx and probability (eX rxmxx`a/ -q wwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwww x are the distance, mass and frequency for the gating coordinate. The role of reorganization and reaction driving force have become essential components in the attempt to deconstruct the role of environmentally coupled protein dynamics required for proton abstraction within a full tunneling model ( 16, 18, 20). For this model, the initial binding mode of the ternary complex for enzyme-bound substrate can undergo further conformational sampling to a position opti mal for catalysis, which is referred to as pre-organization ( 29). The optimization observed through pre-organization deals predominantly with protein dynamics facilita ting the best ground state conformer of the enzyme-bound ligand. Similar to the near att ack conformer (NAC) theory introduced by T.C. Bruice(30, 31), ground state binding modes of substrates in enzymatic reactions 52

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CHAPTER 2: Substrate Pre-Organization in PHM governed by tunneling are evaluated based on wave function overlap relationships between donor and acceptor modes along the tran sfer coordinate. The more efficiently this Michaelis complex (E-S) is formed, the less the solvated protein environment will have to sample the donor-acceptor conforma tions directly affecting the probability of reaching the transition configuration. As shown in figure 2.3 below, the enzymesubstrate (Michaelis complex) has the ability to tune the donor-acceptor distances to reduce the re-organization contribution required to bri ng the reactant and product well into a degenerate state. Within this transition configuration donoracceptor distances at degenerate reactant and product states may require energy (weak interatomic configuration) or may have too much ener gy (strong interatomic configuration) to resemble the optimized equilibrium configuration. The reorganization term will reduce for a binding configuration that approaches the distance associated between donor and acceptor observed at the transition state G). Once the ligand binding position has been achieved, reorganization char acterizes the fine tuning relationship of donor and acceptor distances to attain the optimal de generate energy (de Broglie wave overlap) between enzyme-bound reactant an d product states, respectively( 24, 29, 32). The full tunneling model is completed with the reaction driving force (G) component. This term illustrates the probability of achieving a vibrational state for a through barrier process utilizing thermal activation of a nucleus along its reaction coordinate( 32, 33 ). 53

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CHAPTER 2: Substrate Pre-Organization in PHM Reactant Equilibrium Configuration Product Equilibrium Configuration Transition Configuration Reaction CoordinateFree Energy Equilibrium Configuration Weak Interatomic Configuration Strong Interatomic Configuration Donor Donor Donor Acceptor Acceptor AcceptorReactant Well Reactant Well Reactant Well Product Well Product Well Product Well Reactant Equilibrium Configuration Product Equilibrium Configuration Transition Configuration Reaction CoordinateFree Energy Equilibrium Configuration Weak Interatomic Configuration Strong Interatomic Configuration Donor Donor Donor Acceptor Acceptor AcceptorReactant Well Reactant Well Reactant Well Product Well Product Well Product Well Figure 2.3. Reaction coordinate for a tunneling reaction displaying the distance dependence between donor and acceptor within the transition config uration, denoted as G in figure 2.1, respectively. Previous work by the Klinman group showed the C-H transfer for PHM to occur through a tunneling mechanism( 20). An interesting aspect of these results showed Arrhenius pre-factor and activati on energy of light versus heavy isotope substitution to be both greater and smaller than expected( 20). The authors concluded that a high degree of conformational sampling (gat ing) was present allowing both light and heavy isotopes to tunnel. The current study probes deeper into the mechanisms 54

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CHAPTER 2: Substrate Pre-Organization in PHM underlying these observations by building on the previous work of Wilcox et al( 4 ) and Merkler et al.(5). A further understanding of hydrophobicity in the role of N -acylglycine catalysis was explored to better understand the equili brium processes associated with substrate binding and their effect on C-H cleavage observed in the steady-state. The following study deepens the underlying relationship between protei n dynamics and substrate structure. For our analysis we used a series of non-conjugated, glycine-extended N alkanes to study the primary ki netic isotope effects for C-H bond cleavage with increasing chain length. Theoretical calculati ons were also performed to probe the nature of ternary complex (E-S) behavior using Alchemical free energy perturbation (AFEP) and equilibrium molecular dynamics simulations. AFEP is a molecular dynamics simulation which allows the effect chain length has on the relative dissociation energy from the PHM active site to be determined. The utilization of an active site, hydrophobic pocket and a salt-bridge ligand coordination fo r substrate positioning in the PHM ternary complex was suggested to be the mechanism, by which tuning occurs. The conclusion of this study gives new perspe ctive to the role substrate structure has on preorganization of the PHM reaction. 55

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CHAPTER 2: Substrate Pre-Organization in PHM Materials and Methods: Materials Morpholino-ethane-sulfonic acid (MES), sodium ascorbate, acetyl chloride, propionic anhydride, butyric anhydride, hexanoic anhydride, octanoic anhydride, decanoic anhydride, and cuprous nitrat e were obtained from Sigma, ML, Nacetylglycine (TCI, America), (98%) [-2H2]-glycine was purchased from CDN Isotopes, Triton-X-100 (Fisher), bovine liver catalase (Worthingt on). Recombinant rat PAM was gift from Unigene Laboratories, Inc. (Fairfiel d, NJ, see www.unigene.com). All other experimental reagents were purchased from commercial sources at highest purity grade available and used withou t additional modification. Synthesis of N -Acylglycines, including the -Dideutero-Analogues The N -acylglycines an d the [-2H2]N -acylglycines were synthesized according to the procedures of Wilcox et al .( 4). To a cooled so lution of glycine or [-2H2]-glycine (0 C) and 1.2 equivalents of NaOH, one equiva lent of the corresponding acyl anhydride was added drop wise with stirring. The reaction was allowed to stir for an additional 3 hours with the temperature slowly rising to room temperature and the pH maintained at 78 by the manual addition of NaOH when necessary. An acid extraction of the aqueous phase into EtOAc (3X) was washed with brine then dried over anhydrous MgSO4. Solvent was removed in vacuo the product first recrystallized from a minimum volume of hot ethyl acetate, and then precipitated w ith hexane to yield white crystals. The synthesized N -acylglycines and [-2H2]N -acylglycines were characterized by 400MHz NMR (details listed in the supplemental information). 56

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CHAPTER 2: Substrate Pre-Organization in PHM Steady State Kinetics Reaction Conditions Reactions at 37.0 0.1 C were in itiated by the addition of 0.12-0.18 M PAM (4-5 L) into 2.0 mL of 100 mM MES/NaOH pH 6.0, 30 mM NaCl, 1.0% (v/v) ethanol, 0.001% (v/v) Triton X-100, 1.0 M Cu(NO3)2, 5.0 mM sodium ascorbate, and (0.2 to 10 KM) N -acylglycine (or [-2H2]N -acylglycine). The concentration of dissolved O2 under these conditions was 217 M( 17). Initial rates were measured by following the PAMdependent consumption of O2 using a Yellow Springs Instrument Model 53 oxygen monitor interfaced with a personal computer using a Dataq Instruments analogue/digital converter (model DI-154RS). VMAX,app values were normalized to controls performed at 11.0 mM N -acetylglycine to account for differences in specific activity between different lots of enzyme. Background O2 consumption rates were first determined without enzyme and were subtracted from the rate obtain ed upon PAM addition. Ethanol was added to protect the catalase against as corbate-mediated inactivation( 34) and Triton X-100 was included to prevent nonspecifi c absorption of PAM to the sides of the oxygen monitor chambers. Determination the O 2 -Dependence of the Kinetic Isotope Effects Preliminary experiments to determine which N -acylglycine expressed a kinetic isotope effect were carried out at ambient O2 (217 M) in a non-competitive fashion by comparing the apparent kinetic constants for th e protiated and dideuterated substrates, as described above. The O2-dependence of the kinetic isotope effects expressed by N -acetylglycine was determined by the addition of 0.18 M PAM into 2.0 mL of 100 mM MES/NaOH pH 6.0, 30 mM NaCl, 1.0% (v/v) ethanol, 0.001% (v/v) Triton X-100, 1.0 M Cu(NO3)2, 57

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CHAPTER 2: Substrate Pre-Organization in PHM 5.0 mM sodium ascorbate, 2-50 mM N -acetylglycine (or [-2H2]N -acetylglycine) and 25-830 M O2. The [O2] was varied within the electr ode chamber by mixing different proportions of N2:O2 gas within the headspace above the stirring reaction for 4 minutes. The resulting [O2] was determined from percent saturation observed with the O2 electrode compared to the ambient [O2] as a reference. Background O2 consumption rates obtained before PAM addition were subtracted from those obtained after enzyme was added to initiate the reaction. Viscosity Dependence of the PAM-Catalyzed Oxidation of N -Acylglycines An Ubbelolde viscometer (Industrial Rese arch Glassware Ltd, Union, NJ) size 1B was used to determine the relative kinematic viscosity (rel) of 100 mM MES/NaOH pH 6.0, 30mM NaCl, 1.0% (v/v) ethanol, and 0.001% (v/v) Triton X-100 ( 35). Ten trials were performed in a temperature-controlle d water bath, with viscometer and buffer equilibrated for 10 minutes at 37 0.1 oC. and the values av eraged with corresponding standard deviation. The relative kinematic microviscosity of 100 mM MES/NaOH pH 6.0, 30mM NaCl, 1.0% (v/v) ethanol, and 0.001 % (v/v) Triton X-100 supplemented with sucrose and measured as described for th e non-viscogen control. The experimental, relative kinematic micro -viscosities used were 1(control), 2.08, 3.69, and 5.33 centistokes. The ratio of control : viscogen buffer (0/ was used to compare the VMAX/KM parameters determined for N -acetylglycine and N -decanoylglycine using the rate of oxygen consumption as a probe. Macroviscosity was measured in a similar fashion for both substrates using a Ficoll -400 solution (pH 6.0) to alter the macroviscosity of the reaction environment. The experimental relative kinematic macro58

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CHAPTER 2: Substrate Pre-Organization in PHM viscosity were 1 (control), 3.27, and 5.17 centis tokes. Reactions at 37.0 0.1 C were initiated by the addition of 0.12 M PAM 100 mM MES/NaOH pH 6.0, 30 mM NaCl, 1.0% (v/v) ethanol, 0.001% (v/v) Triton X-100, 1.0 M Cu(NO3)2, 5.0 mM sodium ascorbate, N-acetylglycine or N-decanoylgl ycine and the desired concentration of viscogen (sucrose or Ficoll-type 400). The VMAX,app values were normalized to 11.0 mM Nacetylglycine without added sucrose or Ficoll-400. Analysis of Steady-State Kinetic Data Steady-state kinetic parameters ( standard error) were obtained by a KaleidaGraph fit of the initial velo city (v) vs. initia l substrate concentration ([S]) to the Michaelis-Menton equation (equation 2.5) v = (VMAX,app[S])/(KM,app +[S]) (2.5) where KM,app is the apparent Michaelis constant for the N -acylglycine at fixed [ascorbate] and [O2] and VMAX,app is the apparent maximum velocity at saturating N -acylglycine and fixed [ascorbate] and [O2]. Values for Values for the D(VMAX/KM)app and DVMAX,app were obtained from the quotient of appropriate constants for the protiated N -acylglycine vs. those obtained for the [-2H2]N -acylglycine substrate 1 Initial rate data generated to determine the kinetic mechanism were fit to either a steady-state (equation 2.6) or equilibrium preferred (equation 2.7) mini mal kinetic mechanism using the ENZKIN programs. rate VMAXAG@AO2BC fffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffff KI,AGB KM,O2 KM,O2B AG@A KM,AGB O2BC AG@AO2BC f f f Equation 2.6. Steady-state preferred Michaelis-Menton equation for bi-substrate reactions. 1 Please note, the magnitude of the KIE m easured are a mixture of a primary and -secondary effect deuterium effect. Since the -secondary effect will be negligible (~1. 2) compared to the intrinsic primary deuterium KIE (~10.6), the contributions of this KIE to our measurements are lik ely to be masked within the error ( 12%). 59

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CHAPTER 2: Substrate Pre-Organization in PHM rate VMAXAG@AO2BCKI,AGB K K B AG AG O2fffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffff M,O2M,O2@A@A BC f f f Equation 2.7. Equilibrium preferred Michaelis-Men ton equation for bi-substrate reactions. where AG is the concentration of N -acetylglycine, VMAX represents the maximal velocity for the reaction at infinite concentrations of both O2 and N -acetylglycine, KM values for either substrate are the Michaelis constant at saturating concentrations of the second substrate, and KI, AG is the dissociation constant for Nacetylglycine at a zero [O2]. Kinetic parameters utilizing varying concen tration of one substrate while the other is held constant, for clarity it is important that these term s be introduced and defined: KM,app,AG = Apparent KM for N -acylglycine determined by varying [ N -acylglycine] at fixed [O2] KM,app,O2 = Apparent KM for O2 determined by varying [O2] at fixed [ N -acylglycine] KM,AG = True KM for N -acylglycine determined at saturating concentrations of each substrate KM,O2 = True KM for O2 determined at saturating concentrations of each substrate KI,AG = Dissociation constant for N -acylglycine VMAX,app,AG = Apparent VMAX at saturating [ N -acylglycine] at fixed [O2] VMAX,app,O2 = Apparent VMAX at saturating [O2] at fixed [ N -acylglycine] VMAX = VMAX at saturating [O2] and N -acylglycine (VMAX/KM)APP,AG = Apparent VMAX/KM for N -acylglycine determined by varying [ N -acylglycine] at fixed [O2] (VMAX/KM)APP,O2 = Apparent VMAX/KM for O2 determined by varying [ N -acylglycine] at fixed [ N -acylglycine] 60

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CHAPTER 2: Substrate Pre-Organization in PHM Predicted Docking Conformations of N -Acylglycines within the PHM Active-Site The initial coordinates of the reduced PHM precatalytic complex at 1.85 resolution were obtained from the Protein Data Bank ( http://www.rcsb.org/pdb/ 1SDW) ( 36). Poses were predicted using quantum polarized ligand docking (QPLD) to generate high accuracy substrate binding modes utilizing molecular mechanics (MM) and ab initio programs of the Schrdinger Fi rst Discovery suites, Glide( 37) and Q-site(38 ) respectively ( 39, 40). Initially, Glide is used to select five top poses us ing standard precision (SP) mode. These ligand-receptor complexes are analyzed using the Q-site module where the bound ligand for each selected pose is treated by ab initio methods to calculate partial atomic charges utilizing electrostatic potentia l fitting within the receptor. Displayed within each pose the docked substrate is show n with respect to an active-site hydrophobic pocket Molecular Dynamics using NAMD and Al chemical Free Energy Perturbation i.) Protein Minimization and Equilibration The simulations used a 1.85 resolution X-ray crystal struct ure of residues 43-356 of rat PAM, the PHM catalytic core (PDB, 1SDW) wi th copper domains prepared as listed for AFEP calculations. All molecular dynamics (MD) simulations used the program NAMD 2.6 with the CHARMM22 force fiel d topology treatments for PHM, N -acylglycine, and N -benzoylglycine (hippurate) ( 41-43 ). A water box of 24490 molecules was treated explicitly and parameterized using the TIP3P model, respectively ( 44 ). Minimization and equilibration were both carried out under periodic boundary conditions to eliminate potential surface interactions within the solvent box. Long range electrostatic interactions in this periodic system were treated using the Particle Mesh Ewald sum ( 4561

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CHAPTER 2: Substrate Pre-Organization in PHM 47). The energy minimization protocol for the solvated PHM structure was performed over five consecutive 20 pico second steps; three NVT (cons tant number of particles, volume, and temperature, though no pressure regulation) and two NPT (same as the NVT ensemble with pressure regulation) ensemble steps, respectively ( 48, 49 ). First, in the absence of Langevin dynamics which control pressure, the backbone atoms (C, N, and O) of the PHM were frozen and the remaining por tion of the system (including solvent) was minimized over 10,000 steps. This was a non-backbone atom minimization. Next, the backbone coordinates were released for mini mization of all atoms frozen, followed by another 10,000 step minimization. The final NVT ensemble equilibr ation step involved heating the system with -carbons constrained harmonically for 10,000 steps from 0298.15 K. Next, an NPT ensemble was used to calculate system energetics for system equilibration. The first equilibrati on step applied Langevin piston dynamics to constrained -carbons for system volume equilibration (10,000 steps). The final equilibration repeated the previous step, with the release of all -carbon constraints. The next 100 picosecond step was an NPT-ense mble equilibrium calculation used to determine the unperturbed geometry of the solv ated PHM structure. In this step, the Langevin temperature was set to 310.15 K and the relaxed geometry was attained. Equilibration of the solvated PHM system was determined through the RMSD (root mean square deviation) method. This value, show n versus time, represents spatial movements of the PHM molecule within its solvated envi ronment. Equilibration for the entire system (PHM and water) was determined from flatteni ng of this curve, indicating that motion of PHM within solvent shows little motion fr om the starting coordinates of the PHM (1SDW) crystal structure. 62

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CHAPTER 2: Substrate Pre-Organization in PHM ii.) Alchemical Free Energy Perturbation Method The alchemical free energy perturbation me thod (AFEP) is ideal for the analysis of this type of system as it is a direct probe of chain elongati on effects on dissociation energy of substrates from th e PHM active site. AFEP is a molecular dynamics simulation technique carried out with in the NAMD (NAnoscale Molecular Dynamics) molecular dynamics simulator used to predict the relative energy between two st ates in a system ( 41, 50). This methodology is a dual topology hybrid molecule appr oach used to calculate the free energy difference between two states. The direct transformation between the two thermodynamic states is replaced by a seri es of transformations between non-physical, intermediate states along a pathway. This pa thway is characterized by a variable, referred to as coupling parameter, making the free energy a continuous function of this parameter between A and B. The hybrid Ham iltonian of the system, which is a function of the coupling parameter, which smoothly conn ects state A to state B, is evaluated as shown in equation 2.8 Where Equation 2.8. Expression for the hybrid Hamiltonian for a two state system Where Ha is the Hamiltonian for the initial state, A, Hb is the Hamiltonian for the final state, B, and Ho is the Hamiltonian for the atoms that are not modified during the molecular dynamics simulation. In order to determine the relative free energy differences between substrates, alchemical free energy cal culations were performed on the PHM containing bound 63

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CHAPTER 2: Substrate Pre-Organization in PHM ligand. The force field parameters for both CuH and CuM sites were determined by normal mode analysis in the harmonic approximation. The Hessian was calculated for each Cucontaining (methyl-capped) fragment separa tely, by using Density Functional Theory (DFT)(51). DFT calculations were performe d with the B3LYP hybrid exchangecorrelation functional which included Slater, Hartree-Fock and Becke exchanges as well as the correlation functional of Lee, Yang and Parr (LYP) ( 52-56 ). The effective core and valence electrons within this ab initio treatment were determined with the split valence 613G* basis set. This is a modified vale nce double zeta basis set which is modified containing a polarization function to observe electronic distribution within a nucleus. Equilibrium bond distances, bonding and bending force constants were obtained via geometry optimization. The partial charges of the coppers were those of the formal charges believed to exist based upon th e hypothesized reac tion mechanism and designated Cu(I), respectively. The remaining molecular fragments (i.e. the bound O2, Met-314, His-107, His-108, His-242, and His-244) used potential para meters from the CHARMM22 force field while the glycine ligan d used potential terms selected from the CHARMM22 force field based upon liken ess of chemical environment ( 43). The free energy was calculated using equation 2.9: Equation 2.9 Helmholtz free energy calculation ( A) for utilized for each window (a) in the Alchemical Free Energy Perturbation (AFE P) calculation, respectively. B represents the Boltzmann constant, T is temperature (Kelvin),while b(r,p) and a(r,p)are the Hamiltonians charact eristic of states a and b, respectively. <.>a denotes an ensemble average over configura tions representative of the initial state, a. 64

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CHAPTER 2: Substrate Pre-Organization in PHM The ensemble averaging was performed over the a -th window. In this system, convergence was found to be optimal with the windows divided in inte rvals of 0.1 with the endpoints being sampled every 0.05 to promote convergence. Per window, 10,000 molecular dynamics equilibration steps and 50,000 ensemble averaging steps were performed. The alchemically permutated N -acylglycine substrates were Ndecanoyl-, Noctanoyl-, Nhexanoyl-, Nbutyryl-, Npropionyland Nacetyl-, respectively. Specifically, Ndecanoylglycine was chosen as state = 0 for each trial varying the final permutated ligand state (= 1) to a shorter Nacylglycine derivative. For each ensemble, over each window from =0=1, a thermodynamic integration method averaged was used to estimate Helmholtz free energy change. The variance over each ensemble window was calculated in order to estimate th e error of the derive d values, respectively ( 57). Equilibrium Dynamics Studies of PHM-Substrate Complexes Equilibrium dynamic simulations were performed for all N -acylglycine substrates experimentally tested, as well as an Nbenzoylglycine control. These experiments were conducted to determine the va lidity of the Alchemical Fr ee Energy Perturbation (AFEP) results described above. Experimental setup was identical to the final, unconstrained NPT-ensemble that was used in the final pr otein equilibration step in simulation time (100 ps) and temperature 310.15 K, respectively. The final aspect of the solvated PHM equilibration step was an unrestrained NP T (constant pressure and temperature) molecular dynamics simulation, performed over 1000 picoseconds, in which the relaxed solvated PHM structure determined in the pr ior step was fully equilibrated using the NPT ensemble, respectively. Thr ough the analysis of the equili brium conformations sampled 65

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CHAPTER 2: Substrate Pre-Organization in PHM by each substrate, unconstrained sampling could be compared with the substrate permutation events observed in the AFEP methodology. Each equilibrium dynamics trial was performed using an NPT-ensemble analysis to calculate several total energy values (TOTAL, TOTAL2, and TOTAL3). This was utilized to determine control for errors in kinetic and potential energy term calculations over the 1 nanosecond simulation. TOTAL is the total sum of kinetic energy and potential energy terms, TOTAL2 uses a more highly conserved kinetic energy term for the ensemble calculation, and TOTAL3 is similar to TOTAL2 though with decreased short-time fluctuati ons. The analys is of each energy term allows anomalous state function calculation to be determined by whether the values have large fluctuations from each other. Results Characterization of the Synthe sized N-Acylglycines and [-2H 2 ]N -Acylglycines. [2H2]-N-Acetylglycine. 1H-NMR (400 MHz, Me2SOd6) (singlet, 3H, CH3)singlet, 1H, NH). 13C NMR (100 MHz, Me2SOd6) 172.053 (C=O, carboxylic acid), 170.263 (C=O, amide), 22.877 (CH3, methyl) mp. 205-207oC. [1H2]-N-Propionylglycine. 1H-NMR (400 MHz, Me2SOd6) 0.907 (triplet, J = 7.6 Hz, 3H, CH3, n -alkylchain terminus), 2.066 (quartet, J = 7.6 Hz, 2H, CH2, n-alkylchain methylene linker), 3.670 (singlet, 2H, CH2, -glycine), and 7.955 (singlet, 1H, NH, amide). 13C NMR (100 MHz, Me2SOd6) 174.455 (C=O, carboxylic acid), 172.050 (C=O, amide), 41.180 (CH2, -glycine), 28.815 (CH2, n-alkyl methylene linker), 10.222 (CH3, n-alkyl terminal methyl). mp. 122-124 oC. [2H2]-N-Propionylglycine. 1H-NMR (400 MHz, Me2SOd6) 0.929 (triplet, J = 7.6 Hz, 3H, CH3, n -alkylchain terminus), 2.071 (quartet, J = 7.2 Hz, 2H, CH2, n-alkylchain 66

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CHAPTER 2: Substrate Pre-Organization in PHM methylene linker), and 8.009 (singlet, 1H, NH, amide). 13C NMR (100 MHz, Me2SOd6) 173.940 (C=O, carboxylic acid), 172.099 (C=O, amide), 28.781 (CH2, n-alkyl methylene linker), 10.375 (CH3, n-alkyl terminal methyl). mp. 122-124 oC. [1H2]-N-Butyrylglycine. 1H-NMR (400 MHz, Me2SOd6) 0.804 (triplet, J = 7.4 Hz, 3H, CH3, n-alkylchain terminus), 1.446 (multiplet, 2H, CH2, n-alkylchain methylene linker), 2.035 (triplet, J = 7.2 Hz, 2H, CH2, n-alkylchain methylene linker), 3.675 (singlet, 2H, CH2, -glycine), and 8.052 (singlet, 1H, NH, amide). 13C NMR (100 MHz, Me2SOd6) 172.461 (C=O, carboxylic acid), 171.472 (C=O, amide), 40.526 (CH2, -glycine), 37.001 (CH2, n-alkyl methylene linker), 18.610 (CH2, n-alkyl methylene linker), 13.520 (CH3, n-alkyl terminal methyl). mp. 69-70 oC. [2H2]-N-Butyrylglycine. 1H-NMR (400 MHz, Me2SOd6) 0.717 (triplet, J = 7.4 Hz, 3H, CH3, n-alkylchain terminus), 1.379 (multiplet, 2H, CH2, n-alkylchain methylene linker), 1.950 (triplet, J = 7.2 Hz, 2H, CH2, n-alkylchain methylene linker), and 7.953 (singlet, 1H, NH, amide). 13C NMR (100 MHz, Me2SOd6) 172.594 (C=O, carboxylic acid), 171.548 (C=O, amide), 37.070 (CH2, n-alkyl methylene linker), 18.679 (CH2, n-alkyl methylene linker), 13.681 (CH3, n-alkyl terminal methyl). mp. 69-71 oC. N-Hexanoylglycine. 1H-NMR (400 MHz, Me2SOd6) 0.794 (triplet, J = 7.6 Hz, 3H, CH3, n-alkylchain terminus), 1.240 (multiplet, 4H, (CH2)2, n-alkylchain methylene linker), 1.508 (multiplet, 2H, CH2, n-alkylchain methylene linker),2.086 (triplet, J = 6.8 Hz, 2H, CH2, n -alkylchain methylene linker), 3.744 (singlet, 2H, CH2, -glycine), and 8.049 (singlet, 1H, NH, amide). 13C NMR (100 MHz, Me2SOd6) 173.333 (C=O, carboxylic acid), 172.137 (C=O, amide), 41.237 (CH2, -glycine), 39.695 (CH2, n67

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CHAPTER 2: Substrate Pre-Organization in PHM alkyl methylene linker), 35.727 (CH2, n-alkyl methylene linker), 25.577 (CH2, n-alkyl methylene linker), 22.599 (CH2, n-alkyl methylene linker), 14.556 (CH3, n-alkyl terminal methyl). mp. 88-89 oC. [2H2]-N-Hexanoylglycine. 1H-NMR (400 MHz, Me2SOd6) 0.771 (triplet, J = 6.7 Hz, 3H, CH3, n-alkylchain terminus), 1.171 (multiplet, 4H, (CH2)2, n-alkylchain methylene linker), 1.409 (multiplet, 2H, CH2, n-alkylchain methylene linkers),2.021 (triplet, J = 7.5 Hz, 2H, CH2, n-alkylchain methylene linker), and 7.988 (singlet, 1H, NH, amide). 13C NMR (100 MHz, Me2SOd6) 172.682 (C=O, carboxylic acid), 171.517 (C=O, amide), 35.080 (CH2, n-alkyl methylene linker), 30.884 (CH2, n -alkyl methylene linker), 24.927 (CH2, n-alkyl methylene linker), 21.948 (CH2, n-alkyl methylene linker), 13.875 (CH3, n-alkyl terminal methyl). mp. 88-89 oC. [1H2]-N-Octanoylglycine. 1H-NMR (400 MHz, Me2SOd6) 0.732 (triplet, J = 6.4 Hz, 3H, CH3, n-alkylchain terminus), 1.136 (multiplet, 8H, (CH2)4, n-alkylchain methylene linker), 1.378 (multiplet, 2H, CH2, n-alkylchain methylene linker),1.979 (triplet, J = 7.2 Hz, 2H, CH2, n-alkylchain methylene linker), 3.600 (singlet, 2H, CH2, -glycine), and 7.977 (singlet, 1H, NH, amide). 13C NMR (100 MHz, Me2SOd6) 173.291 (C=O, carboxylic acid), 172.130 (C=O, amide), 41.230 (CH2, -glycine), 39.737 (CH2, nalkyl methylene linker), 35.773 (CH2, n-alkyl methylene linker), 31.912 (CH2, n-alkyl methylene linker), 29.277 ((CH2)2, n-alkyl methylene linker), 22.778 (CH2, n-alkyl methylene linker), 14.618 (CH3, n-alkyl terminal methyl). mp. 103-105 oC. [2H2]-N-Octanoylglycine. 1H-NMR (400 MHz, Me2SOd6) 0.788 (triplet, J = 6.4 Hz, 3H, CH3, n-alkylchain terminus), 1.200 (multiplet, 8H, (CH2)4, n-alkylchain methylene 68

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CHAPTER 2: Substrate Pre-Organization in PHM linker), 1.466 (multiplet, 2H, CH2, n-alkylchain methylene linker),2.073 (triplet, J = 7.2 Hz, 2H, CH2, n-alkylchain methylene linker), and 8.008 (singlet, 1H, NH, amide). 13C NMR (100 MHz, Me2SOd6) 173.037 (C=O, carboxylic acid), 171.670 (C=O, amide), 39.934 (CH2, n-alkyl methylene linker), 35.359 (CH2, n -alkyl methylene linker), 31.518 (CH2, n-alkyl methylene linker), 28.890 ((CH2)2, n-alkyl methylene linker), 25.496 (CH2, n-alkyl methylene linker), 22.368 (CH2, n-alkyl methylene linker), 14.618 (CH3, n-alkyl terminal methyl). mp. 102-104 oC. N-Decanoylglycine. 1H-NMR (400 MHz, Me2SOd6) 0.836 (triplet, J = 6.6 Hz, 3H, CH3, n-alkylchain terminus), 1.238 (multiplet, 12H, (CH2)6, n-alkylchain methylene linker), 1.479 (multiplet, 2H, CH2, n-alkylchain methylene linker),2.079 (triplet, J = 7.4 Hz, 2H, CH2, n -alkylchain methylene linker), 3.702 (singlet, 2H, CH2, -glycine), and 8.076 (singlet, 1H, NH, amide). 13C NMR (100 MHz, Me2SOd6) 173.172 (C=O, carboxylic acid), 172.046 (C=O, amide), 41.733 (CH2, -glycine), 35.678 (CH2, nalkyl methylene linker), 31.920 (CH2, n-alkyl methylene linker), 29.453 ((CH2)3, nalkyl methylene linker),25.806 ((CH2)2, n-alkyl methylene linker), 22.725 (CH2, nalkyl methylene linker), 14.511 (CH3, n-alkyl terminal methyl). mp. 113-114 oC. [2H2]-N-Decanoylglycine. 1H-NMR (400 MHz, Me2SOd6) 0.673 (triplet, J = 6.0 Hz, 3H, CH3, n-alkylchain terminus), 1.058 (multiplet, 12H, (CH2)6, n-alkylchain methylene linker), 1.313 (multiplet, 2H, CH2, n-alkylchain methylene linker),1.919 (triplet, J = 7.2 Hz, 2H, CH2, n-alkylchain methylene linker), and 7.886 (singlet, 1H, NH, amide). 13C NMR (100 MHz, Me2SOd6) 173.249 (C=O, carboxylic acid), 172.080 (C=O, amide), 35.712 (CH2, n-alkyl methylene linker), 31.962 (CH2, n -alkyl methylene 69

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CHAPTER 2: Substrate Pre-Organization in PHM linker), 29.495 ((CH2)3, n-alkyl methylene linker), 25.841 ((CH2)2, n-alkyl methylene linker), 22.759 (CH2, n-alkyl methylene linker), 14.541 (CH3, n-alkyl terminal methyl). mp. 113-114 oC. Steady-State Kinetic Data The (VMAX/KM)app for the PAM-catalyzed oxidation of the N-acylglycines at ambient O2 exhibited a parabolic relationship with the increase in chain length (R = number of carbon atoms in the linear acyl chain). The (VMAX/KM)app increased over 200-fold as the acyl chain lengthened from R = 1 ( N -acetylglycine) to 9 (N decanoylglycine), with the value from 510 50M-1s-1 at R = 1 to (1.3 0.08) 105 M-1s-1 at R = 9, respectively (Fig. 2.4). The increase in the VMAX,app with chain length is only ~1.4-fold, while the KM,app displays a proportionally decr ease as a function of R (~165fold effect) (table 2.1). Th is large increase in the (VMAX/KM)app at ambient O2 represents a decrease in the Nacylglycine concentration required to half-saturate PAM complex at 217 M O2 as described by the Michaelis-Menton equation. These data agree nicely with that reported by Wilcox et al (4). 70

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CHAPTER 2: Substrate Pre-Organization in PHM N -Acylglycine Chain Length (R) 024681 0 (VMAX/KM) (M-1s-1) 0.0 2.0e+4 4.0e+4 6.0e+4 8.0e+4 1.0e+5 1.2e+5 1.4e+5 1 6 e+5 Figure 2.4. The dependence of the VMAX/KM for the PAM-catalyzed oxidation of the N acylglycines on the acyl chain length. The primary deuterium kinetic isotope effect for the C-H bond cleavage for these N acylglycines was decreased linearly as the acyl chain length increased, the D(V/K)app,AG decreasing from 3.21 0.31 at R = 1 to 1.24 0.12 at R = 9, respectively (Fig. 2.5 and table 2.1). VMAX, AMBIENT O2 KM, AMBIENT O2(VMAX/KM) A CYLGLYCINE, AMBIENT O2 D(VMAX/KM)ACYLGLYCINE, AMBIENT O2NameR (s1 ) (mM) (mM1 s1 ) Acetylglycine19.2 (0.3)18.3 (1.6) 0.51 (0.05) 3.21 (0.31) Propionylglycine29.8 (0.3)2.4 (0.3) 2.6 (0.3) 2.94 (0.39) Butyrylglycine310.9 (0.4)2.3 (0.3) 4.6 (0.6) 2.28 (0.33) Hexanoylglycine510.6 (0.2)0.58 (0.05) 18.2 (1.5) 2.13 (0.22) Octanoylglycine713.1 (0.3)0. 20 (0.02) 65.6 (6.1) 1.61 (0.21) Decanoylglycine913.4 (0.2)0.11 (0.01) 126 (8) 1.24 (0.12) Table 2.1 The Non-competitive Deuterium Kinetic Isotope Effect as a Function of N Acylglycine Chain Length. All va lues were determined at 217 M O2. 71

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CHAPTER 2: Substrate Pre-Organization in PHM N -Acylglycine Chain length (R) 024681 0 D (V MAX /K M ) ACYLGLYCINE 0 1 2 3 4 R O N H O OH Parent Structure Figure 2.5. The Decrease in the D(VMAX/KM)app at Ambient O2 as the Acyl Chain Length Increases for PAM Catalysis. Minimal Kinetic Mechanism The N -acylyglycine exhibiting the highest D(VMAX/KM)app,AG at ambient O2 was N -acetylglycine, 3.2 0.31 (table 2.1). The minimal kinetic mechanism for N acetylglycine was determined by measuring the dependence of the D(VMAX/KM)app,AG as a function of O2 concentration. Initial rate data were fit to the bi-substrate kinetic equations representing either the steadyor equilibrium-state preferred mechanisms (equation 2.6 and 2.7), respectively. The data best fit an equilibrium-preferred kinetic mechanism for N -acetylglycine with values of 0.66(H) and 0.40( D) and variance values 0.44(H) and 0.16(D), respectively. By comparis on, data fit to the steady-state preferred kinetic mechanism had comparable and variance values though many calculated parameters were negative and had high erro r suggesting a lack of significance for many 72

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CHAPTER 2: Substrate Pre-Organization in PHM of the terms. The initial rate patterns for both N -acetylglycine and [-2H2]N acetylglycine displayed the unique graph patterns associated with the equilibrium ordered kinetic mechanism: the convergence of 1/initial rate versus 1/[ Nacetylglycine] at increasing [O2] in the second quadrant while the 1 /initial rate versus 1/[O2] at increasing [ N -acetylglycine] intersecting at the abscissa (Fig. 2.6) (17,35,66). Furthermore, an equilibrium random minimal mechanism is differentiated from equilibrium ordered as the replot of (KM,AG/VMAX)app against 1/[O2] inserts at the origin (Fig. 2.6C) ( 17, 58). The resulting kinetic parameters calculated from the equilibrium-preferred kinetic mechanism along with the corresp onding deuterium isotope effect data are shown in table 2.2. 73

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CHAPTER 2: Substrate Pre-Organization in PHM Figure 2,6. Primary and Secondary Plots for N -Acetylglycine (left column) and [-2H2]N -Acetylglycine] (right column). Rows A and B display the Li neweaver-Burke plots for varying [ Nacetylglycine] and [O2]. Rows C and D represents the slope replots from the plots shown in rows A and B. 0 5 10 15 20 25 30 35 0 0.010.020.030.04 1 / [Oxygen] ( M)Slope (KM,AG/VMAX)app 0 2 4 6 8 10 12 14 16 18 0 0.010.020.030.04 1 / [Oxygen] ( M)Slope ( KM,AG MAX app/V ) 0 50 100 150 200 250 300 350 400 450 500 00.10.20.30.40.50.6 1 /[2H2]-N-Acetylglycine (mM)Slope (KM,AG/VMAX)app 0 100 200 300 00.10.20.30.40.50.6 1 / [1H2]-N-Acetylglycine (mM)SlopM,AGMAX)app e (K /V 0 4 8 12 -0.0020.0080.0180.0280.0380.048 1 / [Oxygen] (mM)1 / rate (s1) 0 5 10 15 20 25 -0.0020.0080.0180.0280.0380.0481 / [Oxygen] ( M)1 / rate (s1) -2 2 6 10 14 18 22 26 30 -0.100.10.20.30.40.50.6 1 / [2H2]N -Acetylglycine (mM)1 / rate (s1) 0 2 4 6 8 10 12-0.100.10.20.30.40.50.61 / [1H2] N -acetylglycine (mM) 1 / rate (s1) A B C D 74

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CHAPTER 2: Substrate Pre-Organization in PHM Parameter[1H2] N -acetylglycine[2H2] N -acetylglycine D KIE VMAX 24.9 1.4 22.2 1.41.1 0.1(s-1) (VMAX/KM)OXYGEN36.9 1.9 18.7 0.72.0 0.1(mM-1s-1) KI, N -acet y l g l y cine15.2 0.98 14.4 0.71.1 0.1(mM) Table 2.2. Kinetic parameters for N -acetylglycine and [-2H2]N -acetylglycine from the fit of the kinetic data to rate equation for the equilibrium-preferred kinetic mechanism. The deuterium isotope effects for each relevant kinetic parameter ( standard error) are also included. Consistent with an equilibrium-ordered kinetic mechanism, the magnitude of the D(VMAX/KM)AG and D(VMAX/KM)oxygen terms are equivalent, within experimental error, with the D(VMAX/KM)AG = 1.9 0.23 (table 2.3) and D(VMAX/KM)oxygen = 2.0 0.1 (table 2.2). Replot of the VMAX,app,AG vs. [O2] to determine the D(VMAX) (Fig. 2.7), yielded VMAX values of 21.0 1.6 s-1 for N -acetylglycine and 24.3 2.0 s-1 for [-2H2]N acetylglycine giving a D(VMAX) of 0.88 0.097. These values are in good agreement with those obtained by a fit of the kinetic data to the rate equation for an equilibriumordered mechanism (equation 7): VMAX values of 24.9 1.4 s-1 for N -acetylglycine and 22.2 1.4 s-1 for [-2H2]N -acetylglycine with a D(VMAX) of 1.1 0.1 (tables 2.2 and 2.3). 2 2 The VMAX,app at one fixed [O2] and the D(VMAX/KM)N-acetylglycine value had to be determined from a replot of VMAX,app vs. [O2] because the rate equation for an equilibrium-ordered kinetic mechanism is unsymmetrical. 75

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CHAPTER 2: Substrate Pre-Organization in PHM Figure 2.7. VMAX,app Replot for N -Acetylglycine ( ) and [-2H2]N -Acetylglycine ( ). 76

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CHAPTER 2: Substrate Pre-Organization in PHM [1H2] N -acetylglycine [2H2] N -acetylglycine [Oxygen] VMAX VMAX/KM VMAX VMAX/KM D(VMAX/KM) A G( M)(s-1) SE (mM-1s-1) SE (s-1) SE (mM-1s-1) SE SE 251.0150.0570.0580.0080.3980.0120.0300.0021.9280.300 852.9510.1210.2130.0221.3830.0750.1020.0142.0920.363 3158.9110.1630.6430.0304.5030.1020.4350.0271.4770.115 55011.0370.2731.2420.0897.1700.2190.7360.0621.6890.188 83013.5240.3032.2280.1569.1620.1401.0600.0462.1010.173 Mean1.860.23 Table 2.3. Apparent kinetic parameters for N -acetylglycine and [-2H2]-N -acetylglycine at different initial oxygen concentrations. 77

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CHAPTER 2: Substrate Pre-Organization in PHM It should also be noted, that the D(VMAX/KM)oxygen calculated from re-plot analysis yielded a value of 2.5 0.6 (Fig. 2.7 ). Further analysis of the D(VMAX/KM)AG parameters are listed in table 2.3 and gra phically in figure 2.8. As predicted for an equilibrium ordered mechanism, these data show apparent terms for the D(VMAX/KM)APP,AG kinetic isotope effect to be consta nt as a function of oxygen concentration. Both the dissociation constant for the N -acetylglycine substrate (KI, AG) were shown to be independent of -carbon deuterium substitution, displaying DKI,AG values of 1.1 0.1 and the aforementioned D(VMAX/KM) trends are completely consistent with an equilibrium ordered mechanism (table 2.2)( 59). The kinetic order for the sequential addition of substrates to PAM was determined from primary and secondary re-plots (Fig. 2.6), respectively. According to Cook and Cleland, fo r a bi-reactant system the first enzyme-bound reactant may be determined from the secondary plot trend (KM/VMAX versus 1/substrate) which passes through the origin. Conversely, the second enzyme-bound substrate is determined from the intersection patterns of th e primary plot (1/rate versus 1/substrate) existing at the origin, respectively ( 60). From the plots listed in Fig. 2.6, the primary Lineweaver-Burk ( 61) (double reciprocal/primary) plot of initial rate versus [oxygen] and the secondary plot of slope (KM,AG/VMAX) versus 1/[oxygen] were observed to each intersect at the origin, respectively. 78

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CHAPTER 2: Substrate Pre-Organization in PHM [Oxygen] 0 200 400 600 800 1000 D(VMAX/KM)N -ACETYLGLYCINE 0.0 0.5 1.0 1.5 2.0 2.5 3.0 D(VMAX/KM)ACETYLGLYCINE = 1.86 0.23 Figure 2.8. Replot of the D(VMAX/KM)AG vs. [O2]. Viscosity Effects Values measured for both VMAX and VMAX/KM using the macroviscogen show no deviation from controls for the oxidation of either N -acetylglycine or Ndecanoylglycine, respectively. Increasing the microviscosity w ith sucrose resulted in small decreases in VMAX and KM for N -acetylglycine and N -decanoylglycine. However, the decrease in each value for both N-acylglycines was proportional such that the (VMAX/KM)app,AG showed no dependence on microviscosity (within the lim its of error in the measurements). Specifically, small decreases in VMAX (~2 fold) and KM,APP (~2.5 fold) are observed for both substrates over the relative microviscosity range measured (2.07 5.33 rel). Alterations in the active site microenvir onment were observed to be chain-length 79

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CHAPTER 2: Substrate Pre-Organization in PHM independent as the VMAX/KM term for each substrate was equal and constant over the relative viscosity range studied for both subs trates with non-viscogen controls. This result was a direct comparison of short and long N -acylglycine chain-length under diffusionlimited conditions. The results for th e second-order rate constant of Ndecanoylglycine show little deviation from non-diffusion lim ited values toward theoretical values consistent with a fully diffusion-limited process as determined through the StokesEinstein equation (62) (m =1, figure 2.9). Relative Viscosity ( O/ 012345 6 (VMAX/KM)CONTROL(VMAX/KM)VISCOGEN 0 1 2 3 4 5 6 N -acetylglycine versus microviscosity N -decanoylglycine vesrus microviscosity N -acetylglycine versus macroviscosity N -decanoylglycine versus macroviscosity Theoretical value for a fully diffusion limited Figure 2.9. The effect of the microviscogen (sucrose) and the macro-viscogen (Ficoll-400) on the second order rate constant (VMAX/KM). Relative kinematic viscosities were collected using an Ubbelholde viscometer to determine the viscogenic effect as a function of 100mM MES (pH 6.0) buffer solution standards. Data was fit as the ratio of non-viscogenic control to varying experimental viscosities, respectively. 80

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CHAPTER 2: Substrate Pre-Organization in PHM Chain Length Effect Under Saturating Oxygen Conditions N -acylglycine (AG) RVMAX, SATURATING AG + O2 KM O2, SATURATING AG (VMAX/KM) O2, SATURATING AG Substrate (s1 )( M) (mM1 s1 ) Nacetylglycinea1 24.9 (1.3) 676.8 (70.1) 36.9 (1.9) Nacetylglycineb1 21.0 (0.9) 230 (30) 91.5 (12.0) N -propionylglycine 2 18.7 ( 0.4) 209 (11) 89.7 (5.2) N -butyrylglycine 3 17.1 (0.7) 172 (20) 98.9 (11.9) N -hexanoylglycine 5 20.2 ( 0.6) 147 (14) 137 (13) N -octanoylglycine 7 17.0 (0.3) 107 (7) 158 (11) N -decanoylglycine 9 20.9 (0.6) 102 (11) 204 (23) Table 2.4. Kinetic parameters for (VMAX/KM)OXYGEN determined as a function of N -acylglycine chain length. For N -acetylglycine (VMAX/KM)OXYGEN values were calculated from: (a) the equilibrium preferred equation or (b) saturating [ N -acetylglycine] varying oxygen tension, respectively. The values for (VMAX/KM)APP,O2 displayed a linear effect, increasing proportionally with chain length (figure 2.10). As shown in table 2.4, the (VMAX/KM)OXYGEN increase from 36.9 1.9 mM-1s-1 at N -acetylglycine (R=1) to 204 23 mM-1s-1 N -decanoylglycine (R=9), respectivel y. Interestingly, the mean VMAX = 19.8 0.7 s-1 for all Nacylglycine substrates studied at satura ting concentrations of both oxidizable substrate a nd oxygen, respectively. N -Acyglycine Chain Length (R) 024681 0 (V MAX /K M ) OXYGEN 0 50 100 150 200 250 Figure 2.10. Magnitude of the (VMAX/KM)OXYGEN values measured at saturating concentrations of N acylglycine substrate at ambi ent oxygen concentrations. 81

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CHAPTER 2: Substrate Pre-Organization in PHM Computational Chemistry i.) Predicted Docking Conformations As N -acyl chain length increases within the bi-substrate central complex in PHM, increased interaction with a proposed hydrophobic pocket was observed. This interaction becomes more apparent with increased N -acylglycine chain le ngth. Hydrophobic pocket residues appear to be Met 314, Met 208, Leu 206, and Ile 306, respectively. Of the experimental panel of substrates tested, only N -acetylglycine was not shown to have any interaction with this active-site, hydrophobic domain (f igure 2.14). Within the PHM active site, substrate orienta tion appears to be dependent not only on the interaction between the terminal n-alkane chain and the hydrophobic poc ket, but a second interaction with the guanidino moiety of Arg 240 which forms a bi-dentate salt bridge with the carboxy-terminus of each substrate, except N -acetylglycine. The latter observation is consistent with the co-crystallized substrate, N -acetyl-diiodo-tyrosyl-D-threonine( 63, 64 ), though the postulated hydrophobic pocket has no literature precedence. ii.)Equilibration, Alchemical Free Energy Perturbation (AFEP) and Equilibrium Dynamics The root mean square deviation values for all N -acylglycines and N benzoylglycine were all shown to converge to a constant value. The crystal structure of PHM (1SDW) was resolved to 1.85 respectively( 36). Therefore, this X-ray crystal does not include the position of hydrogen atoms for the protein structure requiring their positions to be determined through an equilibration process ( see protein minimization and equilibration). The RMSD values in figure 2.11 show a small degree of structural distortion from original coordinates of approximately 1.6-1.8 deviation. These values 82

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CHAPTER 2: Substrate Pre-Organization in PHM represent differences directly attributed to thermal fluctuations w ithin the system and suggest that atomic positions in each solvated enzyme-subs trate complex are equilibrated. 0 . . 1 . . 2 020040060080000012001400 Tme (piosecodsRMSD ( ) N -acetylglycine 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 000200300400500600700 Time (picoseconds)RMSD ( ) N -propionylglycine 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 0100200300400500600700 Time (picoseconds)RMSD ( ) N -butyrylglycine 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 0100200300400500600700 Time (picoseconds)RMSD ( ) N -hexanoylglycine 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 0100200300400500600700 Time (picoseconds)RMSD ( ) N -octanoylglycine 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 0100200300400500600700 Time (picoseconds)RMSD ( ) N -decanoylglycine 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 0100200300400500600700 Time (picoseconds)RMSD ( ) N -benzoylglycine 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 020040060080000012001400 Time (picoseconds)RMSD ( ) N -acetylglycine 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 020040060080000012001400 Time (picoseconds)RMSD ( ) N -acetylglycine 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 000200300400500600700 Time (picoseconds)RMSD ( ) N -propionylglycine 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 000200300400500600700 Time (picoseconds)RMSD ( ) N -propionylglycine 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 0100200300400500600700 Time (picoseconds)RMSD ( ) N -butyrylglycine 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 0100200300400500600700 Time (picoseconds)RMSD ( ) N -butyrylglycine 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 0100200300400500600700 Time (picoseconds)RMSD ( ) N -hexanoylglycine 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 0100200300400500600700 Time (picoseconds)RMSD ( ) N -hexanoylglycine 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 0100200300400500600700 Time (picoseconds)RMSD ( ) N -octanoylglycine 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 0100200300400500600700 Time (picoseconds)RMSD ( ) N -octanoylglycine 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 0100200300400500600700 Time (picoseconds)RMSD ( ) N -decanoylglycine 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 0100200300400500600700 Time (picoseconds)RMSD ( ) N -decanoylglycine 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 0100200300400500600700 Time (picoseconds)RMSD ( ) N -benzoylglycine 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 0100200300400500600700 Time (picoseconds)RMSD ( ) N -benzoylglycine Figure 2.11. Converged root mean squared deviation (RMSD) values dete rmined from equilibration of the solvated PHM struct ure (1SDW) for glycine extended substrates. For AFEP calculations, the thermodynamic plot of G versus R shows that relative dissociation energy (GALCHEMICAL) increases as R decreases from 9 to 2. At R = 1, the G values are 12 kcal/mole lower the R = 2 and 4 kcal/mol lower than R = 9, the previous most stable conformation (figure 2.12). The decline in free energy versus chain length indicates that increasing the hydrophobicity of short fatty-acid glycine derivatives compared with Ndecanoylglycine using the Alchemi cal FEP method, causes an increase in the free energy required to decouple the ligand from the central (E-S-O2) complex. Equilibrium dynamics display a drastically altered N -acetylglycine pose compared with all other N -acylglycine substrates. 83

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CHAPTER 2: Substrate Pre-Organization in PHM N -Acylglycine Chain Length (R) 012345678 GALCHEMICAL (kcal/mol) -15 -10 -5 0 5 10 15 Figure 2.12 Alchemical free energy perturbation (AFEP) versus Nacylglycine chain length plot. For the above molecular dynamics (MD) simulations, relative free energies were calculated with N decanoylglycine treated as the initial state ( = 0) with respect to shorter chain N -acylglycine substrates ( =1). Due to the decanoylglycine -to-acetylglycine transformation app earing below the expected trend, butyrylglycine-to-propionylglycine and butyrylglycine-to-acetylglyc ine transformations were also performed and plotted. This NPT simulation attempted to observe the conformational sampling for each substrate in the absence of an alchemical permutation (AFEP). Comparatively, over the 1 nanosecond simulation, N -acetylglycine (R=1) sampled many more binding conformations than any longer substrate, respectively. During this simulation, the N acetylglycine molecule performed an inversio n within the active-site consistent with docking orientation poses predicted in Fig. 2.14. This phenomena was partially observed during the AFEP studies, as the Arg240 salt bridge was not observed at = 1 for the 84

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CHAPTER 2: Substrate Pre-Organization in PHM decanoylglycine-to-acetylglycin e AFEP-transformation, resp ectively. The results displayed sampling behavior id entical to the AFEP simulati on. For the other substrates, the nalkane chain position was modulated by the active-site hydr ophobic pocket and the extent of this interaction decreased proportional to chain length. It should be noted that the N -benzoylglycine equilibrium dynamic (MD) simulation displayed no deviation in the salt bridge between Arg240 a nd the carboxy-terminus of th e glycine moiety. The N benzoylglycine control displaye d a constant Arg240, salt-bridge interaction, similar to Ndecanoylglycine for the entire molecu lar dynamics simulation, respectively. Figure 2.13. Equilibrium dynamic simulations were performed for all N -alkane glycine-extended substrates tested experimentally, along with benzoylglyicne as a control(not graphed). These experiments were conducted to determine the validity of the Alchemical FEP results, through the analysis of equilibrium conformations sampled by each substrate, unconstrain ed compared to that obse rved in the Alchemical methodology. -195 -194.5 -194 -193.5 -193 -192.5 -192 -191.5 -191 -190.5 -190 012345678910 N -acylglycine chain length (R)Helmholtz Free Energy (kcal/mol) TOTAL 1 TOTAL 2 TOTAL 3 The Helmholtz free energy calculation for the equilibrium dynamics simulations showed the energy differences as a function of chai n length to be small over all total energy ensembles (TOTAL 1-3) (Fig. 2.13). Also, the total energies calculated for the N benzoylglycine simulation were observ ed to be within error of the N -acylglycines studied. 85

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CHAPTER 2: Substrate Pre-Organization in PHM N -acetylglycine N -propionylglycine N -butyrylglycine N -hexanoylglycine N -octanoylglycine N -decanoylglycine Figure 2.14. Predicted docking conformations of N -acylglycines within the PH M active site. Poses were predicted using quantum polarized ligand docking ( QPLD ) to generate high accuracy substrate binding modes utilizing molecular mechanics (MM) and ab initio programs of the Schrdinger First Discovery suites, Glide and Q-site respectively. Initially, Glide is used to select five top poses using standard precision (SP) mode. These ligand-receptor complexes are analyzed using the Q-site module where the bound ligand for each selected pose is treated by ab initio methods to calculate partial atomic charges utilizing electrostatic potential fitting within the receptor. Displayed w ithin each pose the docked substrate is displayed in relation to an active-site hydrophobic pocket. The hydrophobic pocket is displayed for clarity as a translucent ora nge mesh with residues omitted to better contrast the n -alkane chain within this region. Atom colors are red (oxygen), blue (nitrogen), grey (carbon), yellow (sulfur), and white (hydrogen), respectively. 86

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CHAPTER 2: Substrate Pre-Organization in PHM Discussion Minimal Kinetic Mechanism from Kinetic Isotope Effects Addition of substrates has been sh own to only occur following the reduction of the two enzyme-bound coppers. The exogenous reductant delivers two electrons from two one-electron oxidation steps converting ascorbate to semi-dehydroascorbate(65). This is not included in the determined minima l mechanism as the reductant is believed to completely dissociate from the active site following reduction with a ping-pong mechanism ( 66-69 ) though the specific reduction fo r the enzyme has not been established. The order of substrate additi on to PAM was determined to be sequential with the addition of N -acylglycine followed by di-oxygen to form the central enzyme complex (E-S-O2). The minimal kinetic mechanis m describing substrate behavior was determined to be equilibrium ordered (Schem e 2.3). This result is consistent with E E-AG E-O2-AG E-(OH)AG-H2OE PRODUCT +k1AG[AG] k2AGk3O2[O2] k4O2k5k7 Scheme 2.3. Representative minimal kinetic mechanism fo r an ordered, sequen tial mechanism for substrate binding to an enzyme. previously detailed kinetic isotope e ffect studies with PAM, respectively ( 16, 17). The rate expression for this minimal mech anism (Equation 10) is unsymmetrical ( 58, 70, 71), resulting in dissimilar reciprocal trends for in itial rate analysis as a function of varied substrate (Fig. 2.6). A signature of th e equilibrium ordered mechanism are the equivalent magnitudes of the D(VMAX/KM) values for N -acetylglycine and oxygen, 1.85 0.86 (Fig. 2.8) and 2.0 0.1 (table 2.1) respectively ( 17, 58, 72). This is due to k2AG being much faster than k3O2[O2] at any [oxygen], respectively ( 17, 60). Therefore the 87

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CHAPTER 2: Substrate Pre-Organization in PHM kinetic isotope effect for this second order ra te constant, are equivalent for each substrate and can be reduced to the following expression: DVMAXKMffffffffffff fgACETYLGLYCINEDk5k5k4ffffff1 k5k4fffffffffffffffffffffffffffDVMAXKMffffffffffff fgOXYGEN Equation 2.10. Detailed expression of the (VMAX/KM) kinetic isotope effect for an equilibrium ordered minimal kinetic mechanism. Therefore, the addition of N -acetylglycine to PAM is expected to bind in an equilibrium fashion, such that its off micro-rate constant ( k2AG) is much greater than micro-rate constants associated with VMAX, ( k5) (Scheme 2.3) ( 17, 59, 60, 71). The experimental evidence provided through the micro-viscosity studies (Fig. 2.9), show no structure dependence on the (VMAX/KM)app,AG parameter for either Nacetylor N -decanoylglycine under ambient oxygen conditions. Therefore, the200-fold increase in catalytic efficiency observed under these conditions c ould not be attributed to a diffusion-limited or partially diffusion-lim ited process (table 2.1) ( 35, 62). This eliminates the possibility that an equilibrium ordered mechanism for N -acylglycine addition to PAM changes to a steady-state preferred mechanis m as chain length increases (17). For a steady-state ordered mechanism, the D(VMAX/KM)AG term would differ from the D(VMAX/KM)O2. The former term would become dependant on th e [oxygen], while the latter would remain constant as a function of [ Nacylglycine] causing the observed KIEs on the second-order rate constant for each substrate to be different ( 71 ). 88

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CHAPTER 2: Substrate Pre-Organization in PHM Interpretation of the V MAX Isotope Kinetic Isotope Effect The results for D(VMAX) (table 2.2) reveal that no tr ansferable proton is available in the rate-limiting step and s uggests that hydrogen transfer is not involved in the ratelimiting step of substrate oxidation. These results are consistent with a model in which the slow catalytic step is formation of a hydroxylating intermediate. In contrast, the experimentally observed D(VMAX) values for the mono-functional domain (PHM) were measured at ~1.5 ( 17, 72) in two separate studies, re spectively. This suggests a difference between the bi-functional (PAM) versus the mono-functional (PHM) domains in which the rate determining step involves a contribution from hydrogen transfer. Further analysis of this phenomenon in PHM was studied with an electron-transfer mutant, in which, a histidine ligand at the CuH domain was mutated to alanine (H172A) ( 72, 73). DVMAXDk5 k5/ k71 k5/ k7 f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f Equation 2.11. Expression of the kinetic isotope effect on VMAX The D(VMAX) values for the mono -functional His172Ala CuH-domain mutant were escalated versus wild-type from ~1.5 to ~3.3, respectively ( 72). The increased D(VMAX) values for wild-type and His172Ala PHM compared with those measured with PAM suggest that C-H bond cleavage is more rate determ ining. Therefore compared to bifunctional PAM, the ratio of k5/ k7 becomes smaller, accentua ting the contribution of the intrinsic net rate constant for bond cleavage within the D(VMAX) value of wild-type and His172Ala PHM, respectively( 71 ). The D(VMAX) term becomes a useful parameter to equate mechanistic trends between PAM, PHM, and DM since its consistently defined for random or ordered as well as steady-state or equilibrium preferred sequential minimal 89

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CHAPTER 2: Substrate Pre-Organization in PHM kinetic mechanisms according to Equation 11 (58, 71 ). The D(VMAX) parameter for DM was observed in the presence of fumarate (an anionic mechanistic activator) to be within error of unity, while increasing to ~2.5 at pH 6.6 in the abse nce of fumarate respectively ( 74). Since the wealth of support fo r electron transfer from the CuH domain is considered to follow C-H bond cleavage ( 16, 72, 75-77 ), one can interpret the difference in the D(VMAX) values between monoand bi-f unctional enzymes to originate from the manner in which electron transfer follows C-H cleavage. Both bi-functional PAM and DM (with fumarate) have a slow er rate of product release ( 17, 74 ) which serves to couple the first irreversible step of C-H bond cleavage to a sequential electron transfer step by increasing the k5/ k7 term, providing a possible e xplanation for the absence of a D(VMAX) value. Therefore, the mono-fu nctional wild-type and His172Ala PHM allow the steps surrounding C-H cleavage, electron transfer and product release to be uncoupled compared with PAM and DM (plus fumarate). This becomes a useful tool for elucidation of the rate determining step in the mechanism of these proteins. From the PHM work, it can be suggested that C-H cleavage is only partially rate determining since the largest D(VMAX) (H172A 3.3) ( 72) value is far below that for the intrinsic net rate constant for C-H cleavage (Dk a value constant for PAM, PHM, and DMFurthermore, the uncoupling of C-H bond cleavage from product release in PHM shows that the substrate hydroxylation/product release steps appear to be rate limiting, not electron transfer. The role of electron transfer from CuH appears to be a requirement for the homolytic cleavage of the copperII-peroxo intermediate (CuII-OOH) to yield the reactive copper hydroxyl 90

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CHAPTER 2: Substrate Pre-Organization in PHM radical species (CuII-O) necessary for radical recombination with the substrate -radical, respectively ( 16, 80). This mechanism agrees explic itly with the Klinman hypothesis, while differs from the mechanism postulated by Chen and Solomons which have hydroxylation followed by electron transfer, respectively ( 16, 75, 81). Structure Dependence on Binding Mode Limitations in the applicability of AF EP theory to PHM (Fig. 2.12) was observed only with calculations containing the transformation to N -acetylglycine (R =1). The cause of this anomalous behavior during th e decanoylglycine-to-ace tylglycine trial can be attributed to the configurational ensemb les not having a large degree of the desired overlap between states, providing requi site accuracy during the permutation( 41). Therefore, the increased substrate chain length (R = 2 9) is directly proportional to increasing dissociation energies of substrate from the ternary complex (Fig. 2.12). Using the AFEP method, the free energy versus chain length plot demonstrates that increasing the hydrophobicity of the Nacylglycine substrate results in a proportional increase of free energy required for ligand decoup ling from the central (E-S-O2) complex. This thermodynamic plot of G versus chain length (R) shows that dissociation energy (GALCHEMICAL) increases as R decreases from 9 to 2. At R = 1, the G values were 12 kcal/mole lower the R = 2 and 4 kcal/mol lower than R = 9, the previous most stable conformation (figure 2.12). These results are derived from the calculation of AFEP values through a direct comparison with decanoylglycine (R = 9). Each substrate is coordinated exactly the same in the central complex (E-S-O2) of PHM, altering only the alkyl chain length. Taking these results in context with all other data, one can suggest that the calculated trend in GDISSOCIATION is true for each substrate (R = 9 2) in its 91

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CHAPTER 2: Substrate Pre-Organization in PHM ternary complex conformation. The therm odynamics can only comment on the relative energy of a conformation once it has been ac hieved, while the kinetics describes the probability of reaching that conformation. Therefore, the increasing chain length promotes alkyl-chain interaction with the proposed hydrophobic pocke t adjacent to the CuM domain (Fig. 2.14). The combination of kinetic and thermodynamic information suggests that the resulting prot ein dynamics which result from this interact ion allow the bound substrate to more efficiently sample conformer distances for optimal wave overlap between C-H and Cu/O2. This observation is most likely a result of an active site extremely well designed to accommodate the spatial requirements for donor and ac ceptor wave overlap. The decrease in the KIE with hydr ophobicity (Fig. 2.5) suggests that environmentally coupled tunneling phenomena become more efficient as the probability of optimal conformer sampling is increased proportional to Nacylglycine hydrophobic poc ket interaction (Fig. 2.14). The decreasing KM,APP for the N -acylglycine substrates with increasing chain length suggests a more facile C-H bond cleavage component proportional to ligand hydrophobicity. Therefore, th e catalytic efficiency (VMAX/KM) of the n-alkane extended substrates is increased while the rate of product release (VMAX) remains constant under saturating conditions of each co -substrate. To account for (VMAX/KM), the chain length dependence for tuning of C-H and CuII-OO wave functions suggest the optimal overlap for acceptor and donor moieties can be regulated by both enzyme dynamics and substrate structure. The constant VMAX value may suggest that a conformational change may be rate-determining for product release. 92

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CHAPTER 2: Substrate Pre-Organization in PHM Previous work on PHM has suggested that both protium and deuterium nuclei in N -benzoylglycine are able to tunnel, as shown by the AH/AD < kH/kD values ( 20). This trend is indicative of an in creased protein gating function responsible for the tunneling reaction. The hallmark features of that study were the temperatureindependent intrinsic kinetic isotope effects above the semi-classical limit while primary kinetic isotope effects displayed temperature dependence allowing deduction of a non-adiabatic full tunneling model ( 20, 28). The equilibrium dynamics run for N -benzoylglycine show a constant bidentate salt bridge interaction with Ar g240, though highly randomized movement of the benzene ring in this substrate was also obser ved. The degree of heavy atom motions are dictated by the magnitude of the AH/AT value (25) and are dominated by the FranckCondon gating term (Equation 4b) ( 32). Therefore, the dependence on distance between donor and acceptor (r) become essential to defining this value. For N -benzoylglycine, the AH/AD value is 5.9 3.2; a very large valu e indicating a large degree of gating ( 20 ). Comparison of the -carbon donor position between both N -benzoylglycine and N decanoylglycine show very little difference in the orientation of these atoms over the course of the MD simulation (see movie files ). This suggests that a decrease in the primary kinetic isotope effect for N -decanoylglycine must deal with the probability of reaching this conformation. The VMAX/KM and KM values for N -decanoylglycine have been observed to be 25-fold higher and 13-fo ld lower (more favorable) than parameters measured for N -benzoylglycine ( 4 ). The role of Arg240 in protein gating has been addressed with the Arg240Gln PHMcc mu tant, with a 2-fold increase in KM and a 200fold decrease in VMAX ( 82). Results from this mutation s uggest that this residue functions to pose a geometrical constraint upon both the ligand and protein allowing the Franck93

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CHAPTER 2: Substrate Pre-Organization in PHM Condon gating dynamics to optimize donor-acceptor distances An advantage for the hydrophobic substrate appears to be an extra st abilization component used to reach the transfer configuration by utilizing the hydrophobic pocket. Therefore, increased contact points of the substrate to the enzyme appear to assist the frequency modulation between both to achieve degener acy more efficiently. Evidence for conformational flexibility Recently, evidence for low frequency conformational sampling was observed experimentally ( 33, 83). The nature of the PHM-coppe r domains were explored using structural techniques (differe ntial scanning calorimetry (DSC) and X-ray crystallography) to study CuM-SMet314 interaction within the CuM domain using the methionine 314 (Met314) to isoleucine (Met314Ile) mutant (83 ). Previous studies on this mutant showed that the thioether inte raction between the methionine 314 ligand and CuM were essential to stabilize the reduced CuI state at CuM domain ( 84). Therefore, upon reduction, the PHM : Cu stoichiometry fell below two as the reduced copper dissociated. The DSC results displayed a decrease in thermostabil ity for the oxidized Met314Ile-PHM mutant versus the wild type structure( 83 ). The difference in thermal denaturation (73C-M314I versus 75.9C-wild type) was assumed to orig inate from the increased lability of bound coppers, in the absence of the CuM-SMet314 ligand. Within this study, the crystal structures for both reduced and oxidized Me t314Ile PHM states were also solved. For the oxidized structure, the release of thioether constraint on CuM displayed an unan ticipated effect; alteration of the CuH-domain geometry affecting histidine (His107) coordination. The reduced crystal structure show ed no bound copper in either CuM or CuH domains, respectively. These results suggested a dua l structural and catal ytic role for PHM 94

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CHAPTER 2: Substrate Pre-Organization in PHM function as both X-ray crystallographic and calorimetric studies point to a structural role for the CuM site, in addition to its establishe d catalytic function. Since X-ray crystallography studies have shown no devi ation in structure between reduced and oxidized PHM ( 36, 64, 83 ), EXAFS has been employed to better resolve the copper ligand fields between oxidation states( 33, 85). Results show CuM-SMet314 interaction to be greatly perturbed by reduction with an incr ease in bond distance from an undetectable distance (>2.5 ) to 2.27 ( 85). The importance to low frequency heavy atom movements was demonstrated by observing changes in CuM-SMet314 shell occupancy as a function of pH ( 33). The shell occupancy in EXAFS is directly correlated to the DebyeWaller (DW) factor, which was used in Bauman et al .( 33 ) to quantify conformational mobility in the CuM domain, respectively. This study gave evidence for previously unknown conformational transiti ons associated with CuM-SMet314 interaction following reduction. Within the reduced state, the CuM-SMet314 DW factor showed pH dependence for modulation, from a decreased "met-on" stat e to an increased "met-off" state. The p Ka for the PHM reaction and the pH dependence of the "met-off" "met-on" transition were both determined to be 5.9, respectively. This tran sition between reduced CuM-SMet314 sub-states was determined to be a global change in the protein st ructure as photoreduction at low temperatures (100 Kelvin, pH 5.1) did not yield the expected "metoff" "met-on" transition ( 33). Since the "met-on" state was inactive, the conformational mobility associated with the "met-off" state appe ars to serve as an indirect probe into the essential role enzyme dynamics cont ributes to the reac tion coordinate. 95

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CHAPTER 2: Substrate Pre-Organization in PHM Source of the Isotope Eff ect in Tunneling Reactions As small perturbations in distance can substantially impact the probability of hydrogen tunneling, as donor and acceptor undergo a close approach, the efficiency of both protium(H) and deuterium(D) transfer is high and the measured KIE can be small. Conversely, when the r increases the smaller wavelength of D leads to a reduced wave function overlap for the heavy isotope, and th e measured KIE can become enormous. As distance increases, the shorter de Broglie wave length attributed to D leads to a reduced wave function overlap and the resulting KIEobs can become several orders of magnitude greater than the semi-classical limit assigned to hydrogen transfer (~7), respectively. In other words, the tunneling probability cha nges as a function of the distance between donor and acceptor. Application of Pre-Organization The data and analysis within this study show the relationship of substrate structure to reaction fidelity. Quantified theoretically with AFEP and experimentally with KIEs, more favorable interaction of the incr easingly hydrophobic substrates containing a carboxy terminus, suggest that the Franck-Condon term can be refined as a function of substrate structure to optimize a central comple x for catalysis. If one considers the strong inter-atomic configuration shown in Fig. 2.3, based on the present findings structurebased design appears well suited to yield a molecular structure having extremely close donor-acceptor contacts with non-glycine extension. This could serve as a novel targeting strategy to develop hi gher affinity analogues of prev iously weak inhibitors. 96

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CHAPTER 2: Substrate Pre-Organization in PHM Conclusion This study gives new perspective to th e role substrate structure has on preorganization of the PHM reaction. The (VMAX/KM)AG increases as a function of chain length due to an increased stabilization of the enzyme-bound ground state. Substrate recognition and tuning appear to be of similar content with in PHM. According to our study, it can be concluded that hydrogen transfer steps are defined by both the environmentally coupled motions as well as th e substrate structure within the PHM active site. The utilization of an active site, hydrophobic pocket and amino-acid salt bridge for substrate positioning in the PHM ternary complex was suggested to be the mechanism, by which, C-H activation became decreasingly rate dete rmining. The role of the active site hydrophobic pocket and Arg240 appear to pre-org anize the enzyme to contribute rate promoting vibrations which assist the through-barrier tunneling process. The decreasing KM,APP for the N -acylglycine substrates with increasing chain length suggests a more facile C-H bond cleavage component proportional to ligand hydrophobicity. Therefore, the catalytic efficiency (VMAX/KM) of the n-alkane extended substrates is increased while the rate of product release (VMAX) remains constant under saturating conditions of each co-substrate. Future work Transition state theory (TST) makes no prediction about how the transition state structure and the free energy of activa tion therein depends on reactant structure( 21). Within TST, the location of the hydrogen species, within the transfer coordinate can be defined at the transition state (TS) by the ma gnitudes of the primary KIEs for the event. Therefore, hydrogen position can determine symm etry within the TS from the magnitude 97

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CHAPTER 2: Substrate Pre-Organization in PHM of the measured KIE. For hydrogen tunneling, KIEs are a function of both the deBroglie wavelength and the extent to which donor an d acceptor orbital overlap occurs. This probability term is much more localized as the proton wavelength shortens and is usually described in terms of a thermally activated vibration along the reacti on coordinate (gating interaction). Once the fluctuating system has attained the transition configuration the tunneling probabilities for the H and D species will be quite different (FC term, Equation 4b) and display an isotope effect on the activated process. Large secondary hydrogen KIEs indicate that the motion of these adjacent atoms is coupled to the reaction coordinate. Correlating substrate structur e to the primary isotope effect, a better understanding for the role ground-state bi nding modes have on the probability of vibrationally excited-state hydrogen transfer was generated from this molecule set. This work may allow direct analysis of secondary isotope effects, well know n to be a probe of tunneling behavior, as C-H can be shown to decrease allowing secondary KIE values to be deduced with increasing chain length ( 86-88 ). Analogous Pre-Organization in D M? The hydrophobic pocket in PHM may have a similar function as an active-site glutamine residue in DM. This is an interesting result in light of the DM studies performed by the Kamichi group who s how the 3,4-dihydroxybenzene moiety of dopamine to have weak-force bonding with Gln369 ( 89, 90). The analogy to the Arg240 salt-bridge in PHM may be explained by the fumarate depe ndent activation ( 74). A convincing hypothesis for the fumarate depend ent activation has this anionic molecule reducing docked phenylethylamine motion through weak bonding inte raction with the ligands primary amine. One manner to better understand similar binding features 98

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CHAPTER 2: Substrate Pre-Organization in PHM between these enzymes may be to study the N -acylglycines using a steered molecular dynamics ( 91) (ex. NAMD) simulation. This study would provide ligand binding trajectories that would resolve the role of n-alkane hydrophobicity has on PHM activesite behavior as final docking conformation was achieved, defining the analogous conclusions to DM would be straightforward. 99

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CHAPTER 2: Substrate Pre-Organization in PHM References: (1) Driscoll, W. J., Chaturve di, S., and Mueller, G. P. (2007) Oleamide synthesizing activity from rat kidney: Identification as cytochrome c. J Biol Chem (2) Merkler, D. J., Merkler, K. A., Ster n, W., and Fleming, F. F. (1996) Fatty acid amide biosynthesis: a possible new role for peptidylglycine alpha-amidating enzyme and acyl-coenzyme A: glycine N-acyltransferase. Arch Biochem Biophys 330, 430-4. (3) Merkler, K. A., Baumgart, L. E., De Blassio, J. L., Glufke U., King, L., 3rd, Ritenour-Rodgers, K., Vederas, J. C., Wilc ox, B. J., and Merkler, D. J. (1999) A pathway for the biosynthesis of fatty acid amides. Adv Exp Med Biol 469, 519-25. (4) Wilcox, B. J., Ritenour-Rodgers, K. J., A sser, A. S., Baumgart, L. E., Baumgart, M. A., Boger, D. L., DeBlassio, J. L., deLong, M. A., Glufke, U., Henz, M. E., King, L., 3rd, Merkler, K. A., Patterson, J. E ., Robleski, J. J., Vederas, J. C., and Merkler, D. J. (1999) N-acylglycine amid ation: implications for the biosynthesis of fatty acid primary amides. Biochemistry 38, 3235-45. (5) Merkler, D. J., Chew, G. H., Gee, A. J., Merkler, K. A., Sorondo, J. P., and Johnson, M. E. (2004) Oleic acid derived metabolites in mouse neuroblastoma N18TG2 cells. Biochemistry 43, 12667-74. (6) King, L., 3rd, Barnes, S., Glufke, U., He nz, M. E., Kirk, M., Merkler, K. A., Vederas, J. C., Wilcox, B. J., and Merkle r, D. J. (2000) The enzymatic formation of novel bile acid primary amides. Arch Biochem Biophys 374 107-17. (7) Shonsey, E. M., Sfakianos, M., Johnson, M., He, D., Falany, C. N., Falany, J., Merkler, D. J., and Barnes, S. (2005) Bile acid coenzyme A: amino acid Nacyltransferase in the amino aci d conjugation of bile acids. Methods Enzymol 400, 374-94. (8) DeBlassio, J. L., deLong, M. A., Glufke U., Kulathila, R., Merkler, K. A., Vederas, J. C., and Merkler, D. J. (2000) Amidation of salicyluric acid and gentisuric acid: a possible role for peptidylglycine alpha-amidating monooxygenase in the metabolism of aspirin. Arch Biochem Biophys 383 46-55. (9) Huitron-Resendiz, S., Sanchez-Alavez, M., Wills, D. N., Cravatt, B. F., and Henriksen, S. J. (2004) Characterization of the sleep-wake patterns in mice lacking fatty acid amide hydrolase. Sleep 27, 857-65. (10) Ritenour-Rodgers, K. J., Driscoll, W. J ., Merkler, K. A., Merkler, D. J., and Mueller, G. P. (2000) Induction of peptidylglycine alpha-amidating monooxygenase in N(18)TG(2) cells: a model for studying oleamide biosynthesis. Biochem Biophys Res Commun 267, 521-6. (11) Hiley, C. R., and Hoi, P. M. (200 7) Oleamide_ A fatty acid amide signaling molecule in the cardiovascular system? Cardiovascular Drug Reviews 25 46-60. (12) Carpenter, T., Poore, D. D., Gee, A. J., Deshpande, P., Merkler, D. J., and Johnson, M. E. (2004) Use of reversed pha se HP liquid chromatography to assay conversion of N-acylglycines to primary fatty acid amides by peptidylglycinealpha-amidating monooxygenase. J Chromatogr B Analyt Technol Biomed Life Sci 809, 15-21. (13) McKinney, M. K., and Cravatt, B. F. (2005) Structure and function of fatty acid amide hydrolase. Annu Rev Biochem 74, 411-32. 100

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CHAPTER 2: Substrate Pre-Organization in PHM (14) Patricelli, M. P., and Cravatt, B. F. (2000) Clarifying the catalytic roles of conserved residues in the amidase signature family. J Biol Chem 275, 19177-84. (15) Patricelli, M. P., and Cravatt, B. F. (2001) Characterizati on and manipulation of the acyl chain selec tivity of fatty acid amide hydrolase. Biochemistry 40, 6107-15. (16) Francisco, W. A., Wille, G., Smith, A. J., Merkler, D. J., and Klinman, J. P. (2004) Investigation of the pathway fo r inter-copper electron transfer in peptidylglycine alpha-amidating monooxygenase. J Am Chem Soc 126, 13168-9. (17) Francisco, W. A., Merkler, D. J., Blackburn, N. J., and Klinman, J. P. (1998) Kinetic mechanism and intrinsic isotope effects for the peptidylglycine alphaamidating enzyme reaction. Biochemistry 37, 8244-52. (18) Klinman, J. P. (2006) The co pper-enzyme family of dopamine betamonooxygenase and peptidylglycine alpha-hydroxylating monooxygenase: resolving the chemical pathwa y for substrate hydroxylation. J Biol Chem 281, 3013-6. (19) Francisco, W. A., Blackburn, N. J ., and Klinman, J. P. (2003) Oxygen and hydrogen isotope effects in an active site tyrosine to phenyl alanine mutant of peptidylglycine alpha-hydroxylating monooxyg enase: mechanistic implications. Biochemistry 42, 1813-9. (20) Francisco, W. A., Knapp, M. J., Bl ackburn, N. J., and Klinman, J. P. (2002) Hydrogen tunneling in peptidylglyc ine alpha-hydroxylating monooxygenase. J Am Chem Soc 124, 8194-5. (21) Shunel, J., and Schowen, R. L. (1991) Theoretical basis for primary and secondary hydrogen isotope effects 1 ed., CRC Press, Inc., Boca Raton, Florida. (22) Marcus, R. A., and N., S. (1985) Electron transfers in chemistry and biology. Biochem. Biophys. Acta 811, 265-322. (23) Knapp, M. J., Rickert, K., and Klin man, J. P. (2002) Temperature-dependent isotope effects in soybean lipoxygenase-1 : correlating hydrogen tunneling with protein dynamics. J Am Chem Soc 124, 3865-74. (24) Nagel, Z. D., and Klinman, J. P. (2006) Tunneling and dynamics in enzymatic hydride transfer. Chem Rev 106, 3095-118. (25) Kohen, A., and Klinman, J. P. (1998) Enzyme catalysis: Beyond classical paradigms. Acc. Chem. Res. 31, 397-404. (26) Meyer, M. P., and Klinman, J. P. (2005) Modelling temperature dependent kinetic isotope effects for hydrogen tr ansfer in a series of soybean lipoxygenase mutants: The effect of anharmonicity upon transfer distance. Chem. Phys. 319, 283-296. (27) Marcus, R. A. (1964) Chemical and Electrochemical Electron-Transfer Theory. Ann. Rev. Phys. Chem. 15, 155-196. (28) Kuznetsov, A. M. a. U., J. (199 9) Proton and hydroge n atom tunneling in hydrolytic and redox enzyme catalysis. Canadian Journal of Chemistry 77, 10851096. (29) Klinman, J. P. (2006) Linking protein st ructure and dynamics to catalysis: the role of hydrogen tunnelling. Philos Trans R Soc Lond B Biol Sci 361, 1323-31. (30) Hur, S., and Bruice, T. C. (2003) The near attack conformation approach to the study of the chorismate to prephenate reaction. Proc Natl Acad Sci U S A 100, 12015-20. 101

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CHAPTER 2: Substrate Pre-Organization in PHM (31) Hur, S., and Bruice, T. C. (2003) Comp arison of formation of reactive conformers (NACs) for the Claisen rearrangement of c horismate to prephenate in water and in the E. coli mutase: the efficiency of the enzyme catalysis. J Am Chem Soc 125, 5964-72. (32) Klinman, J. P. (2006) The role of tunne ling in enzyme catalysis of C-H activation. Biochim Biophys Acta 1757, 981-7. (33) Bauman, A. T., Jaron, S., Yukl, E. T ., Burchfiel, J. R., and Blackburn, N. J. (2006) pH Dependence of peptidyl glycine monooxygenase. Mechanistic implications of Cu-methionine binding dynamics. Biochemistry 45, 11140-50. (34) Chew, G. H., Galloway, L. C., McIntyre, N. R., Schroder, L. A., Richards, K. M., Miller, S. A., Wright, D. W., and Merkle r, D. J. (2005) Ubiquitin and ubiquitinderived peptides as substrates for peptidylglycine alpha-amidating monooxygenase. FEBS Lett 579, 4678-84. (35) Stone, S. R., and Morrison, J. F. ( 1988) Dihydrofolate reductase from Eschichia coli: The kinetic mechanism with NADPH and reduced acetylpyridine adenine dinucleotide phosphate as substrates. Biochemistry 27, 5493-5499. (36) Prigge, S. T., Eipper, B. A., Main s, R. E., and Amzel, L. M. (2004) Dioxygen binds end-on to mononuclear copper in a precatalytic enzyme complex. Science 304, 864-7. (37) GLIDE (2000) Schrodinger, LLC, Portland, OR. (38) QSITE (2000) Schrodinger, LLC, Portland, OR. (39) Friesner, R. A., Banks, J. L., Murphy, R. B., Halgren, T. A., K licic, J. J., Mainz, D. T., Repasky, M. P., Knoll, E. H., Sh elley, M., Perry, J. K., Shaw, D. E., Francis, P., and Shenkin, P. S. (2004) G lide: a new approach for rapid, accurate docking and scoring. 1. Method and a ssessment of docking accuracy. J Med Chem 47, 1739-49. (40) Cho, A. E., Guallar, V., Berne, B. J., and Friesner, R. (2005) Importance of accurate charges in molecular docking: quantum mechanical/molecular mechanical (QM/MM) approach. J Comput Chem 26, 915-31. (41) Phillips, J. C., Braun, R., Wang, W., Gumbart, J., Tajkhorshid, E., Villa, E., Chipot, C., Skeel, R. D., Kale, L., and Schulten, K. (2005) Scalable molecular dynamics with NAMD. J Comput Chem 26, 1781-802. (42) Sanbonmatsu, K. Y., and Tung, C. S. (2006) High performance computing in biology: Multimillion atom simula tions of nanoscale systems. J Struct Biol. (43) MacKerell Jr., A. D., Bashford, D., Bellott, M., Dunbrack Jr., R. L., Evanseck, J. D., Field, M. J., Fischer, S., Gao, J ., Guo, H., Ha, S., Joseph-McCarthy, D., Kuchnir, L., Kuczera, K., Lau, F. T. K., Mattos, C., Michnick, S., Ngo, T., Nguyen, D. T., Prodhom, R., Reiher III, W. E., Roux, B., Schlenkrich, M., Smith, J. C., Stote, R., Straub, J., Watanabe, M ., Wiorkiewicz-Kuczera, J., Yin, D., and Karplus, M. (1998) All-Atom Empirical Potential for Molecular Modeling and Dynamics Studies of Proteins. J. Phys. Chem. B 102 3586-3616. (44) Jorgensen, W. L., Cha ndrasekhar, J., Madura, J. D., Impey, R. W., and Klein, M. L. (1983) Comparison of simple potential functions for simulating liquid water. 1983 79, 926-935. 102

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CHAPTER 2: Substrate Pre-Organization in PHM (45) de Souza, O. N., and Ornstein, R. L. (1997) Effect of pe riodic box size on aqueous molecular dynamics simulation of a DNA dodecamer with particle-mesh Ewald method. Biophys J 72, 2395-7. (46) Cheatham, T. E. I., Miller, J. L., Fox, T., Darden, T. A., and Kollman, P. A. (1995) Molecular Dynamics Simulations on Solvated Biomolecular Systems: The Particle Mesh Ewald Method Leads to St able Trajectories of DNA, RNA, and Proteins. J Am Chem Soc 117, 4193-4194. (47) Essmann, U., Perera, L., Berkowitz, M. L., Darden, T., Lee, H., and Pedersen, L. G. (1995) A smooth particle mesh Ewald method. J. Chem. Phys. 103, 85778593. (48) Melchionna, S. (2000) Constrained systems and st atistical distribution. Phys Rev E Stat Phys Plasmas Fluids Relat Interdiscip Topics 61, 6165-70. (49) English, N. J., and Macelroy, J. M. (2003) Structural and dynamical properties of methane clathrate hydrates. J Comput Chem 24, 1569-81. (50) Humphrey, W., Dalke, A., and Schu lten, K. (1996) VMD: visual molecular dynamics. J Mol Graph 14, 33-8, 27-8. (51) Sousa, S. F., Fernandes, P. A., and Ramos, M. J. (2007) General Performance of Density Functionals. J Phys Chem A (52) Becke, A. D. (1988) Density-func tional exchange-energy approximation with correct asymptotic behavior. Phys Rev A 38, 3098-3100. (53) Johnson, E. R., Dickson, R. M., and Becke, A. D. (2007) Density functionals and transition-metal atoms. J Chem Phys 126, 184104. (54) Lee, C., and Parr, R. G. (1990) Ex change-correlation functional for atoms and molecules. Phys Rev A 42, 193-200. (55) Lee, C., Yang, W., and Parr, R. G. (1988) Development of the Colle-Salvetti correlation-energy formula into a f unctional of the electron density. Phys Rev B Condens Matter 37, 785-789. (56) Becke, A. D. (1993) Density-functiona l thermochemistry. III. The role of exact exchange. J Chem Phys 98, 5648-5652 (57) Beveridge, D. L., and DiCupua, F. M. (1989) Free energy via molecular simulation: apllications to ch emical and bimolecular systems. Ann. Rev. Biophys. Chem. 18, 431-492. (58) Cook, P. F., and Clela nd, W. W. (1981) Mechanistic deductions from isotope effects in multireactant enzyme mechanisms. Biochemistry 20, 1790-6. (59) Rudolph, F. B., and Fromm, H. J. (1979) Plotting inital rate data. Methods in Enzymology 63, 138-158. (60) Cook, P. F., and Cl eland, W. W. (2007) Enzyme kinetics and mechanism Garland Science Publishing, New York. (61) Lineweave, H., and Burk, D. (1934) The determination of enzyme dissociation constants. J. Am. Chem. Soc. 56, 658-666. (62) Brouwer, A. C., and Kirsch, J. F. (1982) Investigation of diffusion-limited rates of chymotrypsin reactions by viscosity variation. Biochemistry 21 1302-1307. (63) Prigge, S. T., Mains, R. E., Eipper, B. A., and Amzel, L. M. (2000) New insights into copper monooxygenases and peptide amidation: structure, mechanism and function. Cell Mol Life Sci 57, 1236-59. 103

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CHAPTER 2: Substrate Pre-Organization in PHM (64) Prigge, S. T., Kolhekar, A. S., Eipper B. A., Mains, R. E., and Amzel, L. M. (1999) Substrate-mediated electron tr ansfer in peptidylglycine alphahydroxylating monooxygenase. Nat Struct Biol 6, 976-83. (65) Merkler, D. J., Kulathila, R., C onsalvo, A. P., Young, S. D., and Ash, D. E. (1992) 18O isotopic 13C NMR shift as pr oof that bifunctiona l peptidylglycine alpha-amidating enzyme is a monooxygenase. Biochemistry 31, 7282-8. (66) Gilligan, J. P., Lovato, S. J., Mehta, N. M., Bertelsen, A. H., Jeng, A. Y., and Tamburini, P. P. (1989) Multiple forms of peptidyl alpha-amidating enzyme: purification from rat medullary thyroid carcinoma CA-77 cell-conditioned medium. Endocrinology 124, 2729-36. (67) Kizer, J. S., Bateman, R. C., Jr., Mill er, C. R., Humm, J., Busby, W. H., Jr., and Youngblood, W. W. (1986) Puri fication and characterizati on of a peptidyl glycine monooxygenase from porcine pituitary. Endocrinology 118, 2262-7. (68) Murthy, A. S., Mains, R. E., and Eipper, B. A. (198 6) Purification and characterization of peptidylglycine al pha-amidating monooxygenase from bovine neurointermediate pituitary. J Biol Chem 261, 1815-22. (69) Li, C., Oldham, C. D., and May, S. W. (1994) NN-dimethyl-1,4phenylenediamine as an alternative reducta nt for peptidylglycine alpha-amidating mono-oxygenase catalysis. Biochem J 300 ( Pt 1) 31-6. (70) Cleland, W. W. (1979) Statistical analysis of enzyme kinetic data. Methods Enzymol 63, 103-38. (71) Cook, P. F. (1991) Kinetic and regulatory mechanism s of enzymes from isotope effects 1 ed., CRC Press, Inc., Boca Raton, Florida. (72) Evans, J. P., Blackburn, N. J., and Klinman, J. P. (2006) The Catalytic Role of the Copper Ligand H172 of Peptidylglyc ine alpha-Hydroxylating Monooxygenase: A Kinetic Study of the H172A Mutant. Biochemistry 45, 15419-15429. (73) Jaron, S., Mains, R. E., Eipper, B. A., and Blackburn, N. J. (2002) The catalytic role of the copper ligand H172 of peptidylglycine al pha-hydroxylating monooxygenase (PHM): a spectrosco pic study of the H172A mutant. Biochemistry 41, 13274-82. (74) Ahn, N., and Klinman, J. P. (1983) M echanism of modulation of dopamine betamonooxygenase by pH and fumarate as dedu ced from initial rate and primary deuterium isotope effect studies. Biochemistry 22, 3096-106. (75) Chen, P., and Solomon, E. I. (2004) O2 activation by binuclear Cu sites: noncoupled versus exchange coupled reaction mechanisms. Proc Natl Acad Sci U S A 101, 13105-10. (76) Chen, P., Bell, J., Eipper, B. A., a nd Solomon, E. I. (2004) Oxygen activation by the noncoupled binuclear copper site in peptidylglycine alpha-hydroxylating monooxygenase. Spectroscopic definition of the resting sites and the putative CuIIM-OOH intermediate. Biochemistry 43, 5735-47. (77) Owen, T. C., and Merkler, D. J. (2004) A new proposal for the mechanism of glycine hydroxylation as catalyzed by peptidylglycine alpha-hydroxylating monooxygenase (PHM). Med Hypotheses 62, 392-400. (78) Miller, S. M., and Klinman, J. P. (1983) Magnitude of intrinsic isotope effects in the dopamine beta-monooxygenase reaction. Biochemistry 22 3091-6. 104

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CHAPTER 2: Substrate Pre-Organization in PHM (79) Takahashi, K., Onami, T., and Noguc hi, M. (1998) Kinetic isotope effects of peptidylglycine alpha-hydroxylating mono-oxygenase reaction. Biochem J 336 ( Pt 1) 131-7. (80) Evans, J. P., Ahn, K., and Klinman, J. P. (2003) Evidence that dioxygen and substrate activation are tightly c oupled in dopamine beta-monooxygenase. Implications for the reactive oxygen species. J Biol Chem 278, 49691-8. (81) Chen, P., and Solomon, E. I. (2004) Oxygen activation by the noncoupled binuclear copper site in peptidylglycine alpha-hydroxylat ing monooxygenase. Reaction mechanism and role of the nonc oupled nature of the active site. J Am Chem Soc 126, 4991-5000. (82) Prigge, S. T., Kolhekar, A. S., Eipper B. A., Mains, R. E., and Amzel, L. M. (1997) Amidation of bioactive peptides: the structure of peptidylglycine alphahydroxylating monooxygenase. Science 278, 1300-5. (83) Siebert, X., Eipper, B. A., Mains, R. E., Prigge, S. T., Blackburn, N. J., and Amzel, L. M. (2005) The ca talytic copper of peptidyl glycine alpha-hydroxylating monooxygenase also plays a cr itical structural role. Biophys J 89, 3312-9. (84) Blackburn, N. J., Rhames, F. C., Ralle, M., and Jaron, S. (2000) Major changes in copper coordination accompany reducti on of peptidylglycine monooxygenase: implications for electron transfer and the catalytic mechanism. J Biol Inorg Chem 5, 341-53. (85) Boswell, J. S., Reedy, B. J., Kulath ila, R., Merkler, D., and Blackburn, N. J. (1996) Structural investiga tions on the coordination envi ronment of the active-site copper centers of recombinant bifuncti onal peptidylglycine alpha-amidating enzyme. Biochemistry 35 12241-50. (86) Tsai, S., and Klinman, J. P. (2001) Probes of hydrogen tunneling with horse liver alcohol dehydrogenase at subzero temperatures. Biochemistry 40, 2303-11. (87) Bahnson, B. J., Colby, T. D., Chin, J. K., Goldstein, B. M., and Klinman, J. P. (1997) A link between pr otein structure and enzyme catalyzed hydrogen tunneling. Proc Natl Acad Sci U S A 94, 12797-802. (88) Pudney, C. R., Hay, S., Sutcliffe, M. J., and Scrutton, N. S. (2006) -Secondary isotope effects as probes of "tunneling -ready" configurations in enzymatic Htunneling: Insight from environm entally coupled tunneling models. J Am Chem Soc 128, 14053-14058. (89) Kamachi, T., Kihara, N., Shiota, Y. and Yoshizawa, K. (2005) Computational exploration of the catalytic mechanism of dopamine beta-monooxygenase: modeling of its mononuclear copper active sites. Inorg Chem 44, 4226-36. (90) Yoshizawa, K., Kihara, N., Kamach i, T., and Shiota, Y. (2006) Catalytic mechanism of dopamine beta-m onooxygenase mediated by Cu(III)-oxo. Inorg Chem 45, 3034-41. (91) Isralewitz, B., Baudry, J., Gullingsrud, J., Kosztin, D., and Schulten, K. (2001) Steered molecular dynamics investigations of protein function. J Mol Graph Model 19, 13-25. 105

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CHAPTER 3: Mechanism of Substrate Hydroxylation 105 Introduction The role of oxygen activation in peptidylglycine -amidating monooxygenase has been the subject of much study[1-5]. The relevance of elucidating the entire oxygen activation process within PAM and its mechanistic sibling, dopamine-betamonooxygenase (D M), is important since they both regulate of vital physiological events. PAM catalyzes the formation of C -terminal carboxamides from their corresponding C -terminal glycines[6-9]. Described in scheme 3.1, amidation reaction of C -terminal glycine extended pro-peptides is considered to be a post-translational modification and is the mechanism used to activate such important neuropeptides as substance P and oxytocin[10-12]. The D M reaction is responsible for the activation of the neurotransmitter norepinephr ine from dopamine[4, 13, 14]. Structures PAM (E.C. 1. 14.17.3) is a bi-functional enzyme cons isting of two heterodomains: Peptidylglycine -hydroxylating monooxygenase (PHM) and peptidylglycine amidoglycolate lyase (PAL). Within PA M, the PHM domain has been the most extensively studied, due to its struct ural and reaction homology to dopaminemonooxygenase (D M)[13, 15]. As a result, both enzy mes share a solvent filled active site separating two essentia l, non-coupled copper atoms[1618]. The PHM X-ray crystal suggests that the two copper domains are as far as ~11 apart [12, 19, 20]. Extended Xray absorption fine structur e (EXAFS) analysis for D M is in agreement with PHM crystallographic data, suggesting that the coppe rs are greater than four angstroms apart [21, 22]. It should be noted that 4 is the critical distance for assembly of the coupled

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CHAPTER 3: Mechanism of Substrate Hydroxylation 106 bi-nuclear 2: 2 bridging geometry of copper with O2 2-[23]. Therefore, the great distance between copper domains ( > 4.0 ) PHM and D M suggest that the active-site bound coppers behave completely as non-bl ue/type-II non-coupled mono-nuclear metal ions. Beyond crystallogra phy, this has been independ ently verified by electron paramagnetic resonance (EPR) and EXAFS studies[7, 16, 24, 25]. The wealth of structural and electronic data have extrem e value, aiding mechanistic resolution when coupled with kinetic analysis [13, 26-28]. The reactions for both PHM and D M involve hydrogen atom transfer from a substrat e/donor complex to an activated Cu/O species, resulting in the stereospeci fic hydroxylation of pro-S -glycine carbon and pro-R benzylic carbon hydroxylation, respec tively (Scheme 3.1a and 3.1b). Peptide O N H HR HS O OH PHM O2H2O2CuII2 ascorbate 2 semidehydroascorbateGLYCINE-EXTENDED PEPTIDEPeptide O N H HR OH O OH PEPTIDE -HYDROXYGLYCINE PALZnII/FeIIIPeptide O NH2 O O OH HO HO NH3 HR HS O2H2O2CuII2 ascorbate 2 semidehydroascorbate D M HO HO NH3 OH HS DOPAMINE NOREPINEPHRINE Scheme 3.1a and 3.1b. Peptidylglycine -amidating monooxygenase (PAM) and dopamine monooxygenase (D M) reactions. In addition to the monooxygenase domain of bi-functional PAM, the PAL domain is a zinc, and recently disc overed calcium and iron-depende nt enzyme which catalyzes de-alkylation of the -hydroxyglycine PHM product to th eir corresponding amide and glyoxylate products (scheme 3.1a) [29-31]. Preci se details of the PAL mechanism have yet to be fully elucidated, though it was broadly considered to catalyze a z inc-hydrolasetype reaction, with rem oval of the substrate C -hydroxyl proton by an active site base

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CHAPTER 3: Mechanism of Substrate Hydroxylation 107 that drove the formation of the glyoxyl ate aldehyde moiety [12, 32]. The irondependence of this reaction was unexpected and is not well understood, though mutation of a conserved tyrosine residue (Tyr 564) eliminates iron binding leading to full inactivation [30]. It was determined th at PAL activity resemble d ureidoglycolate lyase (UGL) (E.C. 4.3.2.3), as both enzymes cataly ze a stereospecific dealkylation of ( S )hydroxyglycine substrates[33, 34]. Unlike UGL, PAL contains a ZnII-FeIII complex with a bridging phenolate (Tyr 564) essential to catalysis[30]. Previous studies that reconstituted PAL with alternate metals (Co+2, Zn+2, Cd+2, and Mn+2) need to be reevaluated in context of this charge-transfer ( =530-570nm; tyrosinate-to-FeIII) complex since several metals (Ca+2, Zn+2, and FeIII) are required for carbinolamide dealkylation[30, 32]. The destination of exogenous metals to apo-PAL, therefore, cannot be considered specific as since several bindin g sites are available during reconstitution. Therefore by utilizing calcium, PAL may rev eal a unique variation of the mixed-valent dinulear complex detailed in purple ac id phosphatase (E.C. 3.1.3.2.), making it an intriguing metallohydrolase[35-38]. The key difference in the reactions catalyzed by PAM and D M is the observed lack of bi-functionality which prevents de-alkylation in D M (scheme 3.1b). The May group (Department of Chemistry, Georgia Ins titute of Technology) has lead the field developing novel substrates a nd characterizing their distinct chemistry for both PAM and D M. For D M sulfoxidation[39], epoxidati on[40], selenoxidation[41], and ketonization[42] oxidations have been reported. The analogous substrate functionalization for PAM is assumed, though of this group of oxidation reactions, only sulfoxidation has been observed [39]. Interestingly, a number of novel D M substrate

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CHAPTER 3: Mechanism of Substrate Hydroxylation 108 analogues demonstrate a de-alkylation proces s similar to that observed with the bifunctional PAM. In D M, N -dealkylation resulted directly from the monooxygenation reaction with N -phenylethylenediamine and N -methylNphenylethylenediamine substrates[43]. Analogous reactions with PAM have also been observed, in which only the PHM domain was utilized for PAL-independent Nand Ode-alkylation reactions. Two examples include; benzylglycine and be nzyloxy acetic acid whic h were oxidized to the benzylamine and benzylalcohol species, with concomitant glyoxylate production for both reactions [31] For PHM and D M, these de-alkylation reactions provide evidence that bond scission results sole ly from the oxidation chemistry of their monooxygenase domains. This similarity in de-alkylation ch emistry provides an interesting framework to study the mechanism of PAM cat alysis [31, 39, 43, 44]. Reaction Mechanism and Model Studies The species presumed responsible fo r hydrogen abstraction in the PHM and D M reactions are predominantly considered to be a copper-superoxo radical species[45]. To date, this important intermediate has not been isolated in either enzyme. Therefore, the modes of copper mediated dioxygen activati on have been probed though computational studies and model studies. Recent contributio ns from the Karlin group (Johns Hopkins University, MD) have prepared end-on/ 1 CuII-superoxo model complexes and shown their ability to orchestrate both H-abstra ction and oxidation chem istry[46-48]. This advance was an exciting confirmation of the previous CuII-superoxo species solved from the PHM crystal structure[19]. The char acterization of a catalytically active 1-CuII-superoxo species has been an essential re sult to this field. Prior to this, both 1 and 2 species had been prepared, though neither exhibited the propensity for both

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CHAPTER 3: Mechanism of Substrate Hydroxylation 109 H-abstraction and oxidation chemistry[49-54] A recent communication illustrated the current paradoxical nature associated with Habstraction and substrat e oxidation. Therein, a CuII-hydroperoxo within a ({tris(2-pyridylmethyl)-amine} TMPA) scaffold was observed to catalyze Nmethyl hydrogen abstraction from a dimethylamine derivative (R-N(CH3)2) and oxidatively Ndealkylate to methyl am ine derivative (R-NH-CH3) and formaldehyde (H2C(O)) major products [46]. Alt hough the characteriza tion of the endon CuII-superoxo species was a critical advance to researchers in the non-coupled binuclear copper-monooxygenase field, the aforementioned example shows that it can not definitively exclude ot her Cu/O nucleophilic or oxida nt species from additional consideration. In the next sections, the present state of oxygen activation in PHM/D M mechanisms will be discussed. Specifically, the CuII superoxo species, both side-on ( 2) and end-on ( 1), will be discussed versus high-valent copper-oxo nucleophiles responsible for H-abstraction. CopperII Superoxo and High-Valent Copper-Oxo Species in PHM/DM The Klinman and Chen/Solomon mechanisms demonstrate the coppersuperoxo nucleophile for substrate activation (scheme 3.2). These mechanisms differ in the initial coordination geometry of the dioxyge n species to copper. At the time of their publication, Chen et al. determined spectroscopic evidence for a side-on/ 2 species from model studies[50, 55]. This L3CuII O2 species was predicted to have an antiferromagnetically c oupled singlet ground state as oppo sed to a closed-shell triplet ground state, respectively [50]. The side-on copperII-superoxo was further concluded by a QM/MM thermodynamic calculation to be the predominant species within the PHM/D M CuM domain compared with the end-on/ 1 analogue [2, 23]. Recently,

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CHAPTER 3: Mechanism of Substrate Hydroxylation 110 evidence for the end-on/ 1 species has been observed in the PHM crystal structure (1.85 ) warranting further study of this species as the nucleophile responsible for C -H abstraction [56]. Both C hen/Solomon and Klinman mechanisms assimilate as an end-on/ 1 CuII-hydroperoxo species is predicted following C -H bond cleavage. The Chen/Solomon mechanism involve s a direct hydroxylation of the C -substrate radical, through a water-assisted radical recombina tion reaction resulting in the simultaneous reduction of the copper-hydroperoxo species and C -OH product release. By water-assisted, the author s refer to the effect coordination of an environmental water molecule ha s on changing the side-on CuII hydroperoxo to an endon moiety. This additional copper ligand is po stulated to adjust th e distal OH of this complex much closer (~1 ) to the substrate-derived radical. Conversely, the CuII-hydroperoxo species in th e Klinman mechanism is reduced through the intramolecular electron transfer from the CuH site yielding a CuII O radical via homolysis. Radical recombination of substrate and Cu/O radical species produce in an inner-sphere alcohol species. Hydrolysis of this intermed iate in the subsequent step releases the hydroxylated product. The Estrin and Yoshizawa mechanisms put forth, instead of a copper-superoxo species, a highly reduced copper oxo species for substrate C H bond cleavage. This rival Cu/O hydrogen-acceptor sp ecies implies a mechanism which un-couples di-oxygen reduction from substrate activation. This reaction is analogous to the ferryl oxidant (FeIV=O) observed with cytochrome P450 during C H activation, referred to as CpI [57, 58]. Through hybrid QM/MM simulations, the CuIII oxide/CuII oxyl species was determined to be thermodynamically preferred compared to CuII-superoxo and

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CHAPTER 3: Mechanism of Substrate Hydroxylation 111 -hydroperoxo counterparts[57, 59, 60]. Both mechanisms propose an environmental modification of the initial end-on/ 1 CuII superoxo species through coupling of an intramolecular electron transfer to the acquisition of two environmental protons to release a water molecule. The resulting CuII oxyl species is treated differently in the Estrin and Yoshiwaza treatments of the reaction coordinate. The Est rin simulation is by far the most computationally rigorous simulation of the theoretical models discussed. This mechanism has a CuII-oxyl species predicted to exis t with two unpaired electrons ferromagnetically coupled to an unpair ed electron delocalized within the CuM ligand domain (Met314, His 107, His 108 L3 +), represented as [L3 +CuM IIIO]2+ or [L3 +CuM IIO-]2+. With a quartet spin ground state of this complex, the H-abstraction event was assumed to be concerted with hydr oxylation of the subs trate radical, though substrate oxidation is accompanied by a spin i nversion from the quartet to doublet ground state. This event couples substrate oxidation directly w ith hydroxylated product release, leaving the L3CuII to bind a water molecule to restore distorted tetrahedral geometry in the oxidized, resting state. The Yos hiwaza mechanism calculated that the CuII oxyl species in its trip let ground state would be the most thermodynamically favorable species for H-abstraction. The following substrate oxidation reaction undergoes a spin inversion to yield an antiferromagnetica lly coupled singlet state to drive con certed H-abstraction with substrate oxidation/product release.

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CHAPTER 3: Mechanism of Substrate Hydroxylation 112R O N H O OH H H CuIICuIIMet His His His His His i) 2eii) O 2CuIICuIMet His His His His His O O Electron transfer Follows C-H cleavage R O N H O OH H CuIICuIMet His His His His His O OH R O N H O OH H CuIICuIMet His His His His His O R O N H O OH H CuIICuIMet His His His His His O OH OH 1 e-R O N H O OH H CuIICuIIMet His His His His His O R O N H O OH H CuIICuIMet His His His His His O R O N H O OH H CuIIMet His His His His His OH 1 e-R O N H O OH H CuIICuIIMet His His His His His OH OH Electron Transfer Precedes C-H Cleavage R O N H O OH H H CuIICuIIMet His His His His His O O H+R O N H O OH H H CuIICuIIMet His His His His His O OH R O N H O OH H H CuIICuIIMet His His His His His O H+ H2O R O N H O OH H CuIICuIIMet His His His His His OH H-Abstraction +2 +2 R O N H O OH H CuIICuIIMet His His His His His OH2 Hydroxylation +2 H2O OH Inital Acti v ationPathforAllPathways Electron Transfer R O N H O OH H CuIICuIMet His His His His His O OH Inner Sphere Alcohol Mechanism H+H2O R O N H O OH H H CuIICuIIMet His His His His His O R O N H O OH H CuICuIIMet His His His His His OH H-Abstraction R O N H O OH H CuIICuIIMet His His His His His Hydroxylation H2O OH Doublet Species Singlet Species Estrin et al. (2006) Yoshizawa et al. (2006) H2O Direct OH Transfer Mechanism Product Release Product Release R O N H O OH H CuIICuIMet His His His His His O OH CuIICuIMet His His His His His O O Chen/Solomon Copper-superoxo Acceptor R O N H O OH H H Klinman Copper-superoxo Acceptor CuIICuIMet His His His His His H2O R O N H O OH H O OH CuIICuIMet His His His His His H2O R O N H O OH H O OH H2O H2O H+, H2O OH2 Electron Transfer Electron Transfer OH2 CuIIOH2 Scheme 3.2 Collection of mechanisms postulated for the hydrogen abstracting and oxidation reactions of peptidylglycine alphaamidating monooxygenase (and dopamine betamonooxygenase), respectively.

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CHAPTER 3: Mechanism of Substrate Hydroxylation 113 Salient Mechanistic Characteristics Of the four postulated mechanisms liste d, a broader analysis of the substrate radical oxidation step shows commonality betw een the Chen/Solomon[23, 61], Estrin and Yoshizawa[59, 60] mechanisms. Thes e mechanisms are similar in that free-product is released as substrate oxidation occurs, with no covalent interaction between H-donor and Cu/O acceptor postulated fo r this process. Of the free product mechanisms, the Estrin and Yoshizawa reaction mechanisms proceed through a concerted C -H cleavage and substrate hydroxylation step. This mechanism was determined to contain an analogous catalyt ic regime to the cytochrome P450 rebound mechanisms [62]. The Chen/Solomon and Yoshizawa mechanisms involve a direct hydroxyl transfer from thei r respective copper/oxygen oxi dant. Conversely, the Klinman mechanism is defined by an inner-sphere alcohol product . This species is produced through a radical recombination reactio n which is then hydrol yzed to yield free -hydroxyglycine product[3, 13]. The direct hydroxyl transfer reaction of the Chen/Solomon and Yoshizawa mechanisms are electronical ly very similar. Each species displays a highly favorable, exotherm ic reaction. Following the spin inversion process required for exothermic hydroxyl transfer, the CuII OH species predicted in the Yoshizawa mechanism transforms into an antiferromagnetically coupled singlet ground state as substrate oxidation pr oceeds. Similarly, the CuII hydroperoxo species proposed by Chen/Solomon has an antiferromagnetically coupled singlet ground state as the most thermodynamically feasible oxidant. Theref ore, as the reaction coordinate proceeds towards substrate oxidation/product release, the transition states of both Yoshizawa Chen/Solomon species become super imposable (scheme 3.3).

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CHAPTER 3: Mechanism of Substrate Hydroxylation 114 The Klinman CuII oxyl radical species is unique in that the re ductive cleavage of the copperII hydroperoxo di-oxygen moiety results from an intra-molecular reduction followed by homolysis to yield the act ive species. Unlike the ferryl (FeIV=O; CpI) complex introduced, the copperII oxyl lacks the strong electron delocalization into the d orbitals of the metal, instead this species can stabilize full radica l character in the 2 orbital of the oxygen[61]. Therefore, the sp ecies could be characterized as either a doublet or a quartet state. The Estrin mechanism postulates a doublet as much more thermodynamically stable than its correspond ing quartet. To further understand this reasoning, it is important to reiterate the mechanism by which the Estrin CuII oxyl oxidant species is formed and how it differs from the Klinman oxidant species. The Estrin CuII oxyl species depends on the delocalization of an electron among the copperdomain ligands to ferromagnetically couple w ith two unpaired electrons within the Cu/O species. Therefore, the H-abstraction occurs in the quartet state, with a spin inversion to the doublet state facilitating an iso-enenergetic oxidation re action step. The Klinman CuII oxyl species forms without the spontaneous addition of solvent protons, instead the CuII superoxo species abstracts the s ubstrate hydrogen to yield the CuII hydroperoxo species. The reduction of this species to the active CuII oxyl radical species is prompted by an intra-molecular electron transfer even t followed by reductive cleavage of the dioxygen species by way of homolysis. This homolytic degradation suggests that the CuII oxyl radical species must exist as a quart et, with well-defined radical character on the oxygen.

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CHAPTER 3: Mechanism of Substrate Hydroxylation 115CuM IIO OH CuM IIHO RC COOH RC COOH Side-on CuII-Peroxo Intermediate CuII-Hydroxyl Intermediate Equivalent Antiferromagnetically Coupled Singlet Ground States Scheme 3.3. Degenerate electronic ground state structures of the CuII-hydroperoxo and CuII-hydroxyl oxidant species proposed for direct substrate oxidation/product release. Please note, copper domain residues were omitted for clarity. Refer to text for more detail. Probing Substrate Oxidation through De-Alkylation As discussed in detail in CHAPTER 1, 18O kinetic isotope effects performed on PHM and D M offer valuable mechanistic information up to and including the O O cleavage step, respectiv ely[58]. These are informative pr obes which can be coupled with C H/D kinetic isotope effect s to postulate the hydrogen abstracting species, though these heavy atom effects are independent of s ubstrate oxidation and pr oduct release steps. Therefore, during the substrate oxidation st ep, differentiation betw een each postulated mechanism becomes complicated to isolate an d distinguish in the absence of a suitable reaction probe (scheme 3.2). The hypothesis of this chapter wa s that using QM/MM methodology, an experimentally well-defined substrate suscep tible to oxidative de-alkylation would serve as a novel probe to determine the Cu/O spec ies responsible for substrate oxidation. Therefore, this study initially involved the design of a no vel PHM substrate able to undergo PAL-independent dealkylation. Th e substrate predicted was benzaldehyde

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CHAPTER 3: Mechanism of Substrate Hydroxylation 116 imino-oxy acetic acid as an analogue to the well-studied PAM substrate, N -benzoylglycine[27, 63, 64]. The imino-oxy mo iety appeared to be a logical dealkylation candidate based on the in situ generation and ESR detection of imino-oxy free radicals [65]. Therefore, following PHM oxida tion the formation of an oxime radical and glyoxylate seemed plausible as a de-alkylation scheme, similar to previously studied dealkylation structures, benzyl glycine and benzyloxyacetic ac id respectively [39]. Modifying the approach of the May group, the nature of the PAL-independent de-alkylation of benzaldehyde imino-oxy acetic acid was analyzed kinetically to ensure that the de-alkylation products released resulted exclusively from a non-enzymatic pathway. Subsequently, a minimal kinetic mechanism for C H cleavage as a function of oxygen was determined for benzalde hyde imino-oxy acetic acid using primary deuterium kinetic isotope effects as the pr obe[27, 63]. Studying the rate-determining chemistry in the presence of a substrate selected for its oxidative instability has the advantage of isolating previously obscure reaction steps present in PHM-dependent C -glycine oxidation. To further st udy the substrate-radical oxidation, in silico analysis using hybrid quantum mechanical/molecular mechanics (QM/MM) calculations were performed on the proposed st ep within the reaction coor dinate. Benzaldehyde imino-oxy acetic acid was used to model free-product generation versus the inner-sphere alcohol radical recombination mechanisms proposed in scheme 3.2. In this study, the experimentally determined product de-a lkylation phenomenon became the probe by which computation selects the copper/oxygen speci es responsible for substrate oxidation. Therefore, the rich chemistry of this novel s ubstrate allows the determination of whether

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CHAPTER 3: Mechanism of Substrate Hydroxylation 117 or not a covalent bond is formed between Cu/O and C -glycyl radical species during the substrate oxidation step of the reaction coordinate. Materials -hydroxyhippurate, benzamide, benzaldehyde oxime, protiated and -dideutero bromo-acetic acid, hydroxylamine hydrochlor ide, along with benzaldehyde were purchased from Sigma-Aldrich. Carboxymethyl hydroxylamine was purchased from TCI. Bovine liver catalase was pur chased from Worthington. N -dansyl-Tyr-Val-Gly was purchased from Fluka Biochimika. All othe r reagents were of the highest quality available from comm ercial suppliers. Recombinant PAM Soluble bi-functional recombinant type A rate medullary carcinoma peptidylglycine -hydroxylating monooxygenase (E.C. 1.14.17.3) was over-expressed in chinese hamster ovary cells and isolat ed from the spent media[66]. Synthesis of Benzaldehyde imino-oxy acetic acid A method for imino-oxy acetic acid synt hesis was employed for the isotopic labeling studies. Benzaldehyde was converted to the corresponding benzaldoxime [67]. The 4.1mmole oxime was dissolved in 20ml of ddH2O and 5 equivalents of NaOH. The reaction was stirred at room temperature for 45 minutes, after whic h 1.5 equivalents of ( -H,H or -D,D) bromo-acetic ac id was slowly added in small increments to the stirring solution of sodium benzaldoximate. Upon completion the reaction as determined by TLC (1:3 Hexanes:EtOAc), the reaction was acidulated with dilute HCl(aq), collected in a Bchner funnel passing petroleum ether over the filtered solid to remove solvent from the filtrate. The white crystalline benzal dehyde imino-oxy acetic acid solid was re-

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CHAPTER 3: Mechanism of Substrate Hydroxylation 118 crystallized twice wi th benzene-petroleum ether. Compound purity was analyzed by RP-HPLC (see Intermediate Stability methods) and 1H and 13C NMR. -di-Protio benzaldehyde imino-oxy acetic acid: 1H NMR analysis (250MHz, MeODd4) 4.96 (singlet, 2H, CH2, -methylene), 7.66-7.87 (m, 5H, ArH), 8.48 (singlet, 1H, H-C=N). 13C NMR analysis (62.5MHz, MeODd4) 171.841 (C=O, carboxylic acid), 149.486 (C=N, imine), 130.911 (Ar, C-1), 129.188 (Ar, C-4), 127.673 (Ar, C-3, C-5), 126.136 (Ar, C-2, C-6), 69.325 (CH2, -methylene), m.p. (93-94C). -di-deutero benzaldehyde imino-oxy acetic acid: 1H NMR analysis (400MHz, Me2SOd6): 7.397-7.589 (m, 5H, ArH), 8.302 (singlet, 1H, H-C=N). 13C NMR analysis (100MHz, Me2SOd6) 171.660 (C=O, carboxylic acid), 150.455 (C=N, imine), 130.865 (Ar, C1), 129.502 (Ar, C-4), 127.688 (Ar, C-3, C-5), 125.644 (Ar, C-2, C-6), m.p. (9697C). Steady State Kinetic Oximetry Initial rates were measured by followi ng the PAM-dependent consumption of O2 using a Yellow Springs Instrument Mode l 53 oxygen monitor wi th a polarographic oxygen electrode interfaced with a personal computer using a Dataq Instruments analogue/digital converter (model DI-154RS) as previously desc ribed in McIn tyre et al. (2006) [68]. Rate of dissolved oxygen disa ppearance was used to determine steady-state values using equation 3.1, as fit to Kaleidagraph.

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CHAPTER 3: Mechanism of Substrate Hydroxylation 119 rate VMAX,appB S@AKM S@A f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f Equation 3.1. Michaelis-Menton equation. Reactions were performed under ambi ent oxygen conditions at 37.0 0.1 C ([O2] = 217 M). Reaction components consisted of 100mM MES/NaOH (pH 6.0), 30mM NaCl, 1% (v/v)EtOH, 0.001% Triton X-100, 10ug/ml bovine liver catalase, 1uM Cu(NO3)2 and 5mM sodium ascorbate. In the pr esence of varying imino-oxy acetic acid substrate, a background rate of oxygen consumption was subtracted from the experimental rate initiated with PAM (80nM). Values for VMAX, app were normalized to published values for 11.0mM N -acetylglycine. Glyoxylate Stoichiometry The stoichiometry of benzaldehyde imino-oxy acetic acid oxidation to glyoxylate formation was determined at 37.0 0.1 o C, 100mM MES (pH 6.0), 30mM NaCl, 1% (v/v) EtOH, 0.001% Triton X-100, 10 g/ml catalase, 1 M Cu(NO3)2, 5mM sodium ascorbate and 5mM benzaldehyde imino-oxy acet ic acid with reactions initiated with 80nM PAM. At specified time points, a 40 L reaction aliquot was removed and quenched with 1% tri-fluoro acetic acid. Conversion of benzaldehyde imino-oxy acetic acid to benzaldoxime was monitored by reverse phase high performance liquid chromatography (RP-HPLC) with a Phenomenex C18-hypersil column using a mobile phase of 65% 50mM KxPO4(pH 6.0), 30% MeOH, and 5% CH3CN flowing at 1mL/min and signal detection at 248nm. A standard curve for the rabbit muscle lactate dehydrogenase (LDH) reduction of glyoxylate to glycolat e was quantified by the oxidation of NADH to NAD+ under experimental quenching conditions for the enzymatic assay [69]. All experimental RP-H PLC samples were diluted to a constant

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CHAPTER 3: Mechanism of Substrate Hydroxylation 120 benzaldoxime concentration of 48 M (standard curve mid point) for glyoxylate analysis. Glyoxylate containing samples were combined with 7.6 units/mL lactate dehydrogenase in KXPO4 (pH 7.0), and 220 M NADH was incubated for 1 hour at 37 0.1 o C then analyzed at A340 with a JASCO V-530 UV-VIS sp ectrophotometer. The ratios of [benzaldoxime] to [glyoxylate] production were represented as thei r respective mean standard deviation calculated for each time point collected. Product Characterization A PAM reaction with 37.0 0.1 o C, 100mM MES (pH 6. 0), 30mM NaCl, 1% (v/v) EtOH, 0.001% Triton X-100, 10 g/ml catalase, 1 M Cu(NO3)2, 5mM sodium ascorbate, and 4mM benzaldehyde imino-oxy acetic acid (BIAA) was initiated with 360nM PAM and left to react for 20 hours. The reaction was diluted with 8mL of ddH2O and extracted with EtOAc (20mL X 3), ri nsed with brine and dried over MgSO4. The product was isolated by flash column chro matography (EtOAc) and concentrated under reduced pressure. To evaluate product purity and polar identity of the isolate a small fraction of the proposed product was also sepa rated for comparison with standards using RP-HPLC analysis while the remaining sample was analyzed by 13C NMR. RPHPLC conditions were as follows: Phenomenex C18-hypersil column using a mobile phase of 65% 50mM KxPO4(pH 6.0), 30% MeOH, and 5% CH3CN flowing at 1mL/min and signal detection at 248nm. Oxygen Stoichiometry Amperometric analysis was used to co rrelate [benzaldehyde imino-oxy acetic acid] to [oxygen] consumed during PAM catalysis. The non-enzymatic background rates of oxygen consumption (Fenton chemistry) were subtracted from rates following PAM

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CHAPTER 3: Mechanism of Substrate Hydroxylation 121 initiation [70, 71]. The concentration of benzaldehyde imino-oxy acetic acid substrate (20, 35, 75, and 100 M) was limiting (below 217 M [O2]) in all trials with reactions performed at 37.0 0.1 o C, 100mM MES (pH 6.0), 30mM NaCl, 1% (v/v) EtOH, 0.001% Triton X-100, 10 g/ml catalase, 1 M Cu(NO3)2, 5mM sodium ascorbate with experimental rates initiated with 1.95nM PAM. The to tal concentration of oxygen consumed as a function of benzaldehyde imino-oxy acetic acid concentration was determined by subtracting the background slope from each point following PAMinitiation. The limit of this function repres ented the total consump tion of substrate and was calculated by fitting the linear portio n of each function to equation 3.2 using SigmaPlot 8.0. The stoichiometry of [i mino-oxy acetic acid] : [oxygen] was expressed as the mean ratio standard deviation fo r the asymptote value (equation 3.2) for each trial, respectively. OxygenBC m B time b Equation 3.2. Asymptote determination used to determine stoichiometric oxygen consumption as slope (m) 0 and y-intercept (b) [Oxygen]. Stability of Putative -Hydroxy-Benzaldehyde Imino-Oxy Acetic Acid A competitive assay was developed to obser ve the kinetic stability of the putative hydroxylated product postulated in the PHMdependent oxidation of benzaldehyde imino-oxy acetic acid. A RP-HPLC separation for N -benzoylglycine, -hydroxyl N -benzoylglycine, benzamide, benzaldehyde imino-oxy acetic acid and benzaldoxime was performed using a Phenomenex C18-hypersil column using a mobile phase of 65% 50mM KxPO4(pH 6.0), 30% MeOH, and 5% CH3CN flowing at a rate of 1mL/min, with signal detection at 248nm. All reactions were performed at 37.0 0.1

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CHAPTER 3: Mechanism of Substrate Hydroxylation 122oC, 100mM MES (pH 6.0), 30mM NaCl, 1% (v/v) EtOH, 0.001% Triton X-100, 5mM sodium ascorbate, and 5.75 g/mL bovine liver catalase, which were brought to a total volume of 500 L. Following initiation with 80nM PAM, 40 L aliquots were removed and immediately quenched with 1% tri-fluoro acetic acid at each specified time point and analyzed with RP-HPLC. The time point ra nge, (including a zero time point) was 0.25 to 100 minutes collected over ten points in duplicate, respectively. For the experimental rates, several concentrations of benzaldehyde imino-oxy acet ic acid (3, 2, and 0.5mM) were reacted in the presence of two concentrations of -hydroxyl N -benzoylglycine (5 and 10mM). Control rates were determined by the rates of product formation from benzaldehyde imino-oxy acetic acid and -hydroxyl N -benzoylglycine in dependently at each specified concentration and time point. Rate analysis for each respective concentration of benzaldehyde imino-oxy acetic acid and -hydroxyN -benzoylglycine were monitored for a decrease in their relative rates of formation against controls. Please note, standard curves for N -benzoylglycine, -hydroxyl N -benzoylglycine, benzamide, benzaldehyde imino-oxy acetic acid and benzaldoxime were prepared under assay conditions with the slope from corresponding peak area values. Non-competitive Isotope Effects -H,H-protiated and -D,D-deuterated benzaldehyde imino-oxy acetic acid were compared independently as a function of oxygen tension. A working stock of benzaldehyde imino-oxy acetic acid was weighed and dissolved into 10mM MES (pH 6.0), and calibrated by correlating the [oxygen] consumption to 20 M Benzaldehyde imino-oxy acetic acid for both derivatives. Mixtures comprised of 100mM MES (pH 6.0), 30mM NaCl, 1% (v/v) EtOH, 0.001% Tr iton X-100, 5mM sodium ascorbate, and

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CHAPTER 3: Mechanism of Substrate Hydroxylation 123 5.75 g/mL bovine liver cat alase at 37.0 0.1 oC were conditioned with varying proportions of an oxygen and nitrogen mixture, with final [oxygen] determined versus ambient oxygen saturation at 37.0 0.1 oC; 217 M respectively[27]. To the chamber headspace, between solution and electrode, th e gas mixture was equilibrated above the stirring solution over four minutes. Appropr iate background rates were measured, with experimental rate initiation using 4-5 L of PAM. Values for both D(VMAX/KM)BIAA,APPARENT and D(VMAX/KM)OXYGEN,APPARENT were attained through fitting of the initial rate da ta to equation 3.1 using Kaleidagra ph, with the ratio of H/D plotted as the mean isotope effect standard deviation. To allow for a more accurate statistical fit of re-plot data, single point D(VMAX/KM)BIAA,APPARENT versus [oxygen] were estimated in duplicate using benzaldehyde imino-oxy acetic acid substrate concen trations below the KM,APPARENT values for each oxygen concentration. This method allowed (VMAX/KM)APPARENT to be estimated directly, as th e observed rate/[s ubstrate] at low [substrate] concentration is expressed in th e units of a second or der rate constant, mM-1s-1 respectively (equation 3.3). Estimation of D(VMAX/KM)BIAA,APPARENT was subsequently performed from the quotient of protiated/ deuterated substrate standard error, respectively rate VMAXB S@AKM S@A f f f f f f f f f f f f f f f f f f f f f f f f f f Q rate VMAXB S@AKM f f f f f f f f f f f f f f f f f f f f f f f f f f ,asS@A<< KM # rate S@A f f f f f f f f f f f VMAXKM f f f f f f f f f f f f f f a s@ 1mM@ 1 Equation 3.3. Manipulation of the Michaelis-Menton equation for single-point VMAX/KM estimation. The plot of D(VMAX/KM)BIAA,APPARENT versus [oxygen] was fit to a hyperbolic curve and analyzed through the least squares method, while the D(VMAX/KM)OXYGEN,APPARENT versus

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CHAPTER 3: Mechanism of Substrate Hydroxylation 124 [benzaldehyde imino-oxy acetic acid] was de scribed as the mean isotope effect standard deviation. Both -di-protiated and -di-deuterated sets (w hich did not include single point D(VMAX/KM)BIAA,APPARENT single point estimations) of data versus varying substrate concentrations were analyzed sepa rately using the program Enzkin and fit to both equations 3.4 and 3.5 within the programs of Cleland[72]. rate VMAXBIAA@AO2BCKI,BIAAB KM,O2 KM,O2B BIAA@A KM,BIAAB O2BC BIAA@AO2BC f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f ff f f f f f f f f f f f f f f f f f f f f f f f f f f Equation 3.4 Steady-state preferred bi-react ant Michaelis-Menton equation. rate VMAXBIAA@AO2BCKI,BIAAB KM,O2 KM,O2B BIAA@A BIAA@AO2BC f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f Equation 3.5. Equilibrium preferred bi-react ant Michaelis-Menton equation. Rates from equations 3.3 and 3.4 are co mprised of substrate concentrations; benzaldehyde imino-oxy acetic acid and oxygen, the maximal initial velocity; VMAX, Michaelis constants; KM,BIAA and KM,O2, and the dissociation constant for substrate A; KI,BIAA. Further data analysis of D(VMAX/KM)BIAA,APPARENT versus [oxygen] and D(VMAX/KM)OXYGEN,APPARENT versus [BIAA] trends were pe rformed to extrapolate values for D(VMAX/KM)OXYGEN,APPARENT as [O2] 0 M, the value of D(VMAX/KM)OXYGEN,APPARENT as [O2] M. The D(VMAX/KM)OXYGEN,APPARENT as [O2] 0 M values were determined through non-linear regression analysis fitting replot data to equation 3.6. The values for D(VMAX/KM)OXYGEN,APPARENT as [O2] M were calculated from the reciprocal e xpression of equation 3.6, shown with the expression in equation 3.7, respectively[73].

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CHAPTER 3: Mechanism of Substrate Hydroxylation 125DVMAXKMf f f f f f f f f f f f f fgBIAA,APPARENT k5Hk5Df f f f f f f fk5H1 k3O2BCk2f f f f f f f f f f f f f f f f f fh j i kk4 k51 k3O2BCk2f f f f f f f f f f f f f f f f f fh j i k f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f1 k5H1 k3O2BCk2f f f f f f f f f f f f f f f f f fh j i kk4 k51 k3O2BCk2f f f f f f f f f f f f f f f f f fh j i k f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f Equation 3.6 Replot data used to fit D(VMAX/KM)BIAA ,as [O2] 0 M according to [74], respectively. 1DVMAXKMf f f f f f f f f f f f f f ffg@DVMAXKMf f f f f f f f f f f f f f ffgO2BCQ 0 M f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f 1DVMAXKMf f f f f f f f f f f f f f ffgO2BCQ 1@DVMAXKMf f f f f f f f f f f f f f ffgO2BCQ 0 M f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f K O2 f f f f f f f 1fg K k2k4 k5 k5 Hbck3k5 k5 Hbc f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f Equation 3.7 Replot data used to fit D(VMAX/KM)BIAA,APPARENT ,as [O2] according to [74], respectively. Quantum Mechanical/Molecular M echanics Reaction Coordinate A QM/MM reaction coordinate s imulation for the PHM-dependent -carbon oxidation of a radical-substrat e allows postulated copper-oxygen species to be evaluated against the experimentally observed de-alk ylation phenomenon. With the assumption that the de-alkylation reaction of benzaldehyde imino-oxy acetic acid benzaldoxime and glyoxylate proceeds through an increase in RC=NO C R bond length, a QM/MM simulation was designed to observe the oxidati on of this substrate. Specifically, the observation of copper-oxygen (Cu O) versus -carbon-oxygen (C O) bond lengths changes were studied as a function of increasing imino-oxy -carbon (C=NO C ) bond length for each postulated species i nvolved for hydroxylation of substrate -carbon radicals (scheme 3.2).

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CHAPTER 3: Mechanism of Substrate Hydroxylation 126 The crystal structure of reduced peptidylglycine -hydroxylating monooxygenase (PHM) was acquired from the protein databank (1sdw.pdb, 1.85 ). All waters were removed from the structure along with all occurrences of the co-crystallized peptide ligand (IYT) outside of the active site[19, 20, 75]. The bond orders and charges were then corrected on the co-crystallized protein structure. The copper atoms atom types were designated as CuI, respectively. With no furthe r energy minimizations of the cocrystallized prot ein structure, ProteinPrep was used to add hydrogens and perform a restrained minimization. The bounding box containing all ligand atoms was increased from 10 3 (default) to 14 3. The molecular oxygen was removed to prepare the structure using the ProteinPrep wizard to prepare for the quantum polarized ligand docking (QPLD) docking of benzaldehyde im ino-oxy acetic acid. Poses were predicted using the quantum polarized ligand docking ( QPLD ) module to genera te high accuracy substrate binding modes utilizing molecular mechanics (MM) and ab initio programs of the Schrdinger First Discovery suites (www. schrodinger.com), Glide[76] and Q-site[77] respectively [78-82]. Initially, Glide[76] was used to select five top poses using standard precision (SP) mode. These ligand-receptor co mplexes were analyzed using the Q-site module where the bound ligand for each selected pose was treated by ab initio methods to calculate partial atomic charges utilizi ng electrostatic potential fitting within the receptor. QPLD was performed using defa ult settings with receptor and van der Waals scaling set to 1.0 and 0.8 respectively, except for the quantum mechanical level which was set to accurate instead of fast. Po se selection was determined through the rejection of poses exceeding the following sel ected values for root mean squared (RMS) and maximum atomic displacement, 0.5 and 1.3 respectively. Single-point energy

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CHAPTER 3: Mechanism of Substrate Hydroxylation 127 determination was treated by the QPLD algorithm to determine the most energetically favorable ligand pose with respec t to the receptor. Glide was then used to re-dock the ligand using each of the ligand charge sets prev iously calculated in Q-site[77]. To the QPLD output pose with the benzaldehyde imi no-oxy acetic acid substr ate docked into the PHM crystal structure, 3 Na atoms were added to neutralize (zero ne t charge) the system along with the molecular oxygen using the c oordinates from the original pdb. The system was then solvated using the TIP3P water model (explicit solvation) and all waters beyond 10 angstroms from the structure were removed. The coordinates for the protein/benzaldehyde imino-oxy acetic acid/Cu/O2 complex were then frozen and the system was minimized for 3000 steps. Impos ed constraints on the frozen atoms were then released followed by another 3000 step minimization of the system. Alpha-carbons were then constrained and O2 was frozen and the system wa s then heated from 100 200 degrees Kelvin over 20 picoseconds in 1 femp tosecond time steps. Then the same thing was done again from 200 310.15 degrees Kelv in over another 20 picosecond (ps). Then one 50 ps equilibrium dynamics run wa s performed under constant pressure and temperature (NPT) for volume equilibration of the system. The constraints were then released on the alpha-carbons a nd the entire system equilibrated for 500,000 ps. It should be noted that the O2 was frozen during the entire syst em equilibration because it was not formally bound to the CuM domain and would therefore tr averse the active site during equilibration if its coordinate s were not held constant. Th e final minimized structure of the PHM-ligand central complex was attained through quantum mechan ical treatment of the CuM pocket while treating the remainder of th e protein structure classically to yield the correct final geometry. With all constr aints released for this analysis, the QM/MM

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CHAPTER 3: Mechanism of Substrate Hydroxylation 128 system calculations were intended to allow the solvated central complex to find its most favorable and energetically minimized ground state structure. The resulting structure obtained from this QM/MM initial minimizatio n calculation was used with Q-Site[77] for our reaction coordinate calcula tions. For the QM portion of the calculation a spin unrestricted hybrid density functional theo ry, with B3LYP (Beck e-style-3-parameter density functional theo ry with the Lee-Yang-Parr correl ation functional) hybrid-exchange functional with an LAVCP* (combination of 6-31G* and Lanl2Dz ba sis sets) to more accurately define copper atoms using effectiv e core potentials within the basis set [8386]. The molecular mechanics portion of th e protein was treated with the OPLS_2001 force field of Jorgensen[87, 88]. Due to th e presence of radical in the system, the quantum mechanical region was treated with a spin unrestricted open-shell calculations (UDFT). There were two species of interest fo r study in the PHM oxidation reaction: The copper-hydroxyl (CuII OH) and the copper-oxyl radical (CuII O). From the work by Crespo et al. [57], it was determined that the CuII OH species could be studied with either the singlet or triplet spin state, while the CuII O species could be observed with doublet or quartet spins. The CuII OH species in the singlet spin state was previously constructed by Crespo et al .[57]. Therefore, the CuII OH singlet state (free product ) was chosen to compare with the Cu-alkoxide species which was CuII O with quartet spin (inner-sphere alcohol product ). Following the equilibration process described above, CuII-superoxo species and the bound ligand were set to CuII OH or CuII O and benzaldehyde imino-oxy acetic acid -carbon radical in the input file, respectively. The quantum mechanical (QM) region was calcula ted using the Cu atom and domain residues

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CHAPTER 3: Mechanism of Substrate Hydroxylation 129 (His240, His242, Met314), the metal-bound oxygen species (either OH or O), and benzaldehyde imino-oxy acetic acid C radical substrate. On th is selection, a Q-site job was run without coordinate constraints to determine th e optimized starting point geometry. The calculation was re-done w ith the oxygen species frozen and the C of benzaldehyde imino-oxy acetic acid frozen to determine the energetic minima starting point. Analysis of the reacti on coordinate for substrate oxidation was determined through the difference calculated from incremental increase in the distance between the copper bound oxygen bond and the correspondi ng decrease in the C O(H) bond. The coordinates determined for each change in re action coordinate were re-frozen for those two atoms and the energy calculated quantum mechanically. Results were visualized using the Maestro [89] Results Reaction Stoichiometry, Product Iden tification and PH M Product Stability 13C NMR product analysis shows a spectrum w ith similar chemical shifts to that of the benzaldehyde imino-oxy acetic acid starting material, containing signature aromatic and imine carbon shifts (repres ented A-E, 3.1). Though, corresponding to the O acetyl moiety, the product spectrum was missing two carbon shifts at 171.8 and 69.3 ppm, respectively. The isolated product wa s observed to have a retention time of 12.4 minutes which differed from that observed for the benzaldehyde imino-oxy acetic acid starting material, 6.0 minutes respectively, using the RP-HPLC conditions listed in the methods section.

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CHAPTER 3: Mechanism of Substrate Hydroxylation 130 Figure 3.1. 13C NMR analysis of PAM mediated benzaldehyde imino-oxy acetic acid catalysis. The isolated sample was analyzed with a Varian 100-MHz instrument using ( d3)-methanol as the solvent. Product analysis was further analyzed using the modified lactate dehydrogenase assay for the conversion of glyoxylate to glycolate (scheme 3.4) and RP-HPLC for benzaldoxime, quantification of the glyoxylat e : benzaldoxime ratio formed by PAM was determined to be 1.05 0.14 (figure 3.2). Scheme 3.4. Spectrophotometric glyoxylate analysis used in tandem with C18 RP-HPLC for the determination and dilution benzaldoxime concentration to 48M, respectively. D C B A D C B A D C B A N OH C A A B B D E E

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CHAPTER 3: Mechanism of Substrate Hydroxylation 131 Figure 3.2. Determination of the PAM dependant ratio of formed products (oxime and glyoxylate) using the imino-oxy acetic acid substrate. The concentration of the oxime product was determined for each time point by RP-HPLC, the samples were then diluted to a constant concentra tion for spectrophotometric glyoxylate analysis. Using benzaldehyde imino-oxy acetic acid, the stoichiometry of PAM dependant oxygen consumption with initial substrate concentration was shown to be 0.97 0.06 (figure 3.3 and table 3.1). 0 20 40 60 80 100 120 Time [Oxygen] ( M) 0 20 40 60 80 100 120 Time [Oxygen] ( M) 100 M 75 M 35 M 20 M 0 20 40 60 80 100 120 Time [Oxygen] ( M) 0 20 40 60 80 100 120 Time [Oxygen] ( M) 100 M 75 M 35 M 20 M 0 20 40 60 80 100 120 Time [Oxygen] ( M) 0 20 40 60 80 100 120 Time [Oxygen] ( M) 100 M 75 M 35 M 20 M 0 20 40 60 80 100 120 Time [Oxygen] ( M) 0 20 40 60 80 100 120 Time [Oxygen] ( M) 100 M 75 M 35 M 20 M 0 20 40 60 80 100 120 Time [Oxygen] ( M) 0 20 40 60 80 100 120 Time [Oxygen] ( M) 100 M 75 M 35 M 20 M 0 20 40 60 80 100 120 Time [Oxygen] ( M) 0 20 40 60 80 100 120 Time [Oxygen] ( M) 100 M 75 M 35 M 20 M Table 3.1 and Figure 3.3. Stoichiometry of the enzymatic [oxygen] versus [substrate] consumption measured below the ambient concentration of dissolved oxygen (217uM). Benzaldehyde imino-oxy acetic acid was used in this experiment as the substrate. 0 0.5 1 1.5 2 020406080100Time (minutes)Concentration [mM] Benzaldoxime Glyoxylate

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CHAPTER 3: Mechanism of Substrate Hydroxylation 132 Stability of the Benzaldehyde IminoOxy Acetic Acid Oxidation Product Following substrate oxidation via the PH M domain of bi-functional PAM, there are two possible pathways for oxime product ge neration. The first, displayed in scheme 3.5 as Catalytic Path A, represents a stable -hydroxy-benzaldehyde imino-oxy acetic acid product which requires the PAL domain of bi-functional PAM to enzymatically catalyze the benzaldoxime and glyoxylate pr oduct formation. Catalytic Path B represents product formation from the PAM reac tion to require only the oxidation step of the monooxygenase domain of the bi-func tional enzyme, PHM. Under these circumstances, the oxidation of the -carbon would be followed by a PAL-independent, chemical de-alkylation to yield products (scheme 3.5). N O O OH N OH O OH O Benzaldehyde IminoOxy Acetic Acid Benzaldehyde Oxime GlyoxylatePHM 2asc, O22sda, H2O 2CuII N O O OH OH H -Hydroxy-Benzaldehyde Imino-Oxy Acetic Acid PAL ZnIINon-Enzymatic Dealkylation C a t a l y t i c P a t h AC a t a l y t i c P a t h B Scheme 3.5. Representation of the two possible pathways for benzaldoxime and glyoxylate formation following benzaldehyde imino-oxy acetic acid oxidatio n in the PHM-domain. Pl ease note that asc and sda represents ascorbate and semi -dehydroascorbate, respectively. Using initial rate kinetics, competitive dealky lation was used to probe the stability of the proposed -hydroxy-benzaldehyde imino-oxy acetic acid intermediate versus -hydroxyhippurate, a known PAL s ubstrate (scheme 3.5b).

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CHAPTER 3: Mechanism of Substrate Hydroxylation 133 O N H OH O OH O NH2 O H O OH Benzamide Glyoxylate N OH O OH O Benzaldehyde Oxime Glyoxylate PAL ZnIINon-Enzymatic DealkylationC a t al y t i c P a t h AC a t a l y t i c P a t h B Known PAL Substrate -Hydroxy-Hippuratei f C a t a l y t i c P a t h A N OH O NH2 i f C a t a l y t i c P a t h B = ca. 1 for both Experimental and ControlsRates of Formation of BOTH N OH AND O NH2 will decrease versus controls Experimental Variable Scheme 3.5b. Experimental design to kinetica lly differentiate the two possible degradation pathways for benzaldehyde imino-oxy acetic acid to its corresponding benzaldoxime product following PHM-dependent oxidation. A reverse-phase-HPLC separation of react ants and products for this experiment (represented with figure 3.4) was used to cal culate conversion of both benzaldehyde imino-oxy acetic acid to benzaldoxime and -hydroxyN -benzoylglycine to benzamide. Figure 3.4. C18 -reverse phase high performance liquid chromatograph of (R/S)--hydroxyN benzoylglycine, N -benzoylglycine, benzamide, benzaldehyde imino-oxy acetic acid, and benzaldoxime using 65% 50mM KxPO4(pH 6.0), 30% MeOH, and 5% CH3CN flowing at a rate of 1mL/min, with signal detection at 248nm.

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CHAPTER 3: Mechanism of Substrate Hydroxylation 134 The rates of benzaldoxime formation obser ved in the presence of either 6mM or 10mM -hydroxyN -benzoylglycine compared with cont rols display rates which appear undifferentiated over the range of benzaldehyde im ino-oxy acetic acid and -hydroxyN benzoylglycine concentrations, respectively (figure 3.5). Figure 3.5. Time course for the PAM-dependent conversion of 3, 2, and 0.5 mM benzaldehyde imino-oxy acetic acid to benzaldoxime in the presence and absence of -hydroxyN -benzoylglycine. Results were calculated as a function of percentage conv ersion determined from RP-HPLC analysis. Figure 3.6, displays the reciprocated e ffect shown in figure 3.5; the effect conversion of benzaldehyde imino-oxy acetic ac id to benzaldoxime has on the conversion of -hydroxyN -benzoylglycine to benzamide. Figure 3.6 Resolved time course for the conversion of 6mM and 10mM -hydroxyN -benzoylglycine to benzamide in the presence and absence of 0.5, 2, and 3 mM benzaldehyde imino-oxy acetic acid (BenIAA), respectively. Time (minutes) 0246 [Benzamide] (mM) 0 2 4 6 8 10mM -OH Hipp / No BenIAA 10mM -OH Hipp / 3mM BenIAA 10mM -OH Hipp / 2mM BenIAA 10mM -OH Hipp / 0.5mM BenIAA Time (minutes) 0123456 [Benzamide] (mM) 0 1 2 3 4 5 6mM -OH Hipp / No BenIAA 6mM -OH Hipp / 3mM BenIAA 6mM -OH Hipp / 2mM BenIAA 6mM -OH Hipp / 0.5mM BenIAA ime (minutes) 24 2 [ OH Hippurate] (mM) 2 4 mM eAA mM mM eAA mM 2mM eAA mM mM eAA ime (minutes) 24 2 [ OH Hippurate] (mM) 2 4 mM eAA mM mM eAA mM 2mM eAA mM mM eAA Time ( minutes ) 020406080100120 [Benzaldoxime] (mM) 0 1 2 3 4 3mM BenIAA / No -OH Hipp 3mM BenIAA / 10mM -OH Hipp 3mM BenIAA / 6mM -OH Hipp Time ( minutes ) 020406080100120 [Benzaldoxime] (mM) 0.0 0.5 1.0 1.5 2.0 2.5 2mM BenIAA / No -OH Hipp 2mM BenIAA / 10mM -OH Hipp 2mM BenIAA / 6mM -OH Hipp Time ( minutes ) 020406080100120 [Benzaldoxime] (mM) 0.0 0.2 0.4 0.6 0.8 0.5mM BenIAA / No -OH Hipp 0.5mM BenIAA / 10mM -OH Hipp 0.5mM BenIAA / 6mM -OH Hipp

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CHAPTER 3: Mechanism of Substrate Hydroxylation 135 With respect to PAM dependant dealkylation rates, results suggest that benzaldoxime versus benzamide formation display constant rates for both experimental and control conditions which observed product formation se parately as well in parallel. Kinetic Isotope Effects D(VMAX/KM) analysis of BIAA oxida tion show a decline in D(VMAX/KM)BIAA as O2 0uM, while D(VMAX/KM)OXYGEN remains constant over the range of constant BIAA concentrations (tab les 3.2 and 3.3). Table 3.2. Kinetic parameters for data in figures 3.7 and 3.8, respectively. Table 3.3. Kinetic parameters for oxygen variation with benzaldehyde imino-oxy acetic concentration held constant. Data fit to the steady-stat e preferred mechanism (equation 3.4) had root square values ( ) of 0.237 and 0.140 fo r protiated and -dideuterated benzalde hyde imino-oxy acetic acid, respectively. Conversely, data fit to the equilibrium preferred mechanism

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CHAPTER 3: Mechanism of Substrate Hydroxylation 136 (equation 3.5) displayed root square values ( ) of 0.236 and 0.156 for the protiated and -dideuterated species. The major difference in the statistical fitting of the data occurred observing the average residual l east square parameter, referre d to as variance[72]. An approximate ten-fold increase in this term was observed with the equilibrium preferred mechanism, as compared with its steady-st ate preferred counterpart. The values for protium and deuterium compounds incr eased from 0.0561(H) and 0.0230(D) from equation 3.4 to 0.555(H) and 0.242 (D) in equa tion 3.5, respectively. This large increase in the average residual least square (variance) term represents a greater deviation in error between experimental rates (VEXP) and the pr edicted/optimized rates (VOPT). Kinetic data fit to the steady-stat e preferred model (equation 3.2) displayed an increased statistical significance based on their varian ce terms even though the square root ( ) are statistically very similar. The graphs of 1/rate versus 1/[BIAA] and 1/rate versus 1/[Oxygen] both show intersections in the same quadrant, with a ne gative abscissa and positive ordinate when fit to the steady-state preferred equation, re spectively. The extrapolated value of D(VMAX/KM)BIAA as[O2] 0uM was determined to be 14.65 0.95 (figure 3.7) and the D(VMAX/KM)BIAA as [O2] was calculated to 1.01 0.03 (figure 3.8).

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CHAPTER 3: Mechanism of Substrate Hydroxylation 137 0 2 4 6 8 10 12 14 16 02004006008001000D(VMAX/KM)BIAA[Oxygen] ( M) Figure 3.7. Replot analysis of data represented in table 3.2 for the D(VMAX/KM)BIAA.

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CHAPTER 3: Mechanism of Substrate Hydroxylation 138 -0.4 -0.35 -0.3 -0.25 -0.2 -0.15 -0.1 -0.05 010203040501 ----------------------------------------------------------------------[(VMAX/KM)H/(VMAX/KM)D] [(VMAX/KM)H/(VMAX/KM)D] (O2 ---> [0])1 / [Oxygen] ( Figure 3.8. Replot analysis of data represented in table 3.2 for the D(VMAX/KM)BIAA as[O2] 0uM and D(VMAX/KM)BIAA as [O2] respectively. The non-competitive isotope effects for be nzaldehyde imino-oxy acetic acid oxidation range in magnitude from within error of unity for D(VMAX/KM)BIAA as [O2] deviating from an aforementioned finite value as [O2] 0 M, 14.7 1.0. Table 3.4. Kinetic parameters determined from the steadystate preferred bi-substrate Michaelis-Menton equation 3.4, respectively.

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CHAPTER 3: Mechanism of Substrate Hydroxylation 139 The magnitude of the D(VMAX/KM)OXYGEN was calculated from equation 3.4 to a value of 5.78 0.71 (table 3.4) This value agrees with individually obtained experimental values, showing a finite and constant value over several [BIAA] versus varying [oxygen] of 4.69 1.08 (figure 3.9, blue circles, O ). Figure 3.9 The dependence of [oxygen] on D(VMAX/KM)BIAA ( BLACK ) and the [Benzaldehyde iminooxy acetic acid] dependence on D(VMAX/KM)O2 ( BLUE ). D(VMAX) was also observed to be n ear unity at 0.84 0.08 and the D(KI,BIAA) was observed to have an isotope effect of 2.06 0.52. The KM,BIAA parameters for both protiated and di-deuterated spec ies were determined to have negative values with large standard errors, suggesting this term may not be present due to lack of statistical significance in that term[27, 72]. QM/MM Reaction Coordinate The two species chosen for study were the CuII OH (singlet) and the CuII O (quartet). The potenti al energy of the reaction coordinate for the CuII OH

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CHAPTER 3: Mechanism of Substrate Hydroxylation 140 singlet species, which was calculated as the difference in bond distance between the C O and Cu OH bonds, displayed an essentially smooth increase in relative energy from the starting point to a saddle point value of +45.93 kcal/mol. Following this point, the calculated energy of the reaction coordi nate decreased to -25.98 kcal/mol. The C O bond lengths decrease 4.60 to 2.80 at the energetic saddle point, resting at a final distance of 1.30 respectively. The CuII OH bond lengths were observed to increase over the reaction coordinate from 1.82 through 2.81 at the energetic maxima, to 4.08 at the energetic minima, respectively. The imino oxy carbon bond lengths for benzaldehyde imino-oxy acetic acid was observed to change 0.16 over the reaction coordinate, from 1.36 to 1.52 respectively. The sequence shown in sche me 3.6, represent the quantum mechanical portion of the QM/MM study performed. The displayed poses corre spond directly with their respective steps in the figure 3.10, respectively. The s ubstrate exhibits a constant orientation of the carboxy-term inus of the benzaldehyde imi no-oxy acetic acid substrate until the step prior to the saddle point (+37.45 kcal/mol). Following this point, a coupled rotation in the hydroxyl moiety and the carboxy te rminus of the substrate is shown which orients the substrate -carbon essentially perpendicula r to the approaching hydroxyl group (scheme 3.6).

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CHAPTER 3: Mechanism of Substrate Hydroxylation 141 Reaction Coordinate [(C -O) (Cu-OH)] -3-2-10123 Potential Energy (kca/mol) -40 -20 0 20 40 60 (NO-C Bond Dinstance (Angstrom) 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 (Cu-OH) RC vs G (Cu-OH) RC vs NO-C Bond Dist. Figure 3.10. QM/MM simulated reaction coordinate for the oxidation chemistry observed for the benzaldehyde imino-oxy acetic acid C-radical oxidation with a singlet CuII-OH species (circles). The squares represent de-alkylation distances observed for imino-oxy C bond lengths versus reaction coordinate.

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CHAPTER 3: Mechanism of Substrate Hydroxylation 142 Scheme 3.6. CuM domain for the PHM active site poses for benzaldehyde imino-oxy acetic acid C-radical for the reaction coordinate corresponding to figure 3.10, respectively. Yellow encased coordinate represents the energetic maxima. Atomic representations are carbon (grey), nitrogen (blue), sulfur (yellow), oxygen (red), and hydrogen (white). Pleas note, the yellow bordered box represents the energetic maxima for the reaction coordinate.

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CHAPTER 3: Mechanism of Substrate Hydroxylation 143 The resulting reaction coordinate from the copper-alkoxide (CuII O.) derivative (figure 3.11) show the potential energy trend to be less ordered than that observed with the cupric-hydroxyl species. Sp ecifically, there is an ener getic plateau midway to the saddle point at ~28 kcal/mol, respectively. Following this, the sa ddle point is observed to be at +54.75 kcal/mol, which was +12.82 k cal/mol greater than the value observed for the cupric-hydroxyl species reaction coordina te, respectively. Although the potential energy was observed to be greater for the cupr ic-oxyl radical species, the potential energy versus reaction coordinate trend decreased dr amatically faster than the cupric-hydroxyl, respectively. The cuprous hydroxyl potential energy versus reaction coordinate (RC) decreased from +45.93 kcal/mol at RC = 0.014 to -25.97 kcal/mol at RC = 2.78 while the cupric-oxyl radical decreased from +58.75 kcal/mol at RC = 1.78 to -9.68 kcal/mol at RC = 3.05, respectively. Theref ore, the total change in potential energy for each species was within error of each other for each species, 68.43 kcal/mol (CuII O) and 71.90 kcal/mol (CuII OH).

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CHAPTER 3: Mechanism of Substrate Hydroxylation 144 Reaction Coordinate [(C -O)-(Cu-O)] -3-2-10123 Potential Energy (kcal/mol) -20 0 20 40 60 80 NO C Bond Distance (Angstrom) 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 (Cu-Alkoxide) RC vs G (Cu-Alkoxide) RC vs NO-C Bond Dist. Figure 3.11. QM/MM simulated reaction coordinate for the oxidation chemistry observed for the benzaldehyde imino-oxy acetic acid C-radical oxidation with a quartet CuII-O species (circles). The squares represent de-alkylation distances observed for imino-oxy C bond lengths versus reaction coordinate.

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CHAPTER 3: Mechanism of Substrate Hydroxylation 145 Scheme 3.7 PHM active site poses for benzal dehyde imino-oxy acetic acid C-radical for the reaction coordinate corresponding to figure 3.11, respectively. The yellow coordinate represents the energetic maximum. Atomic representations are carbon (grey), nitrogen (blue), sulfur (yellow), oxygen (red), and hydrogen (white). Please note, the yellow bordered box represents the energetic maximum in the reaction coordinate.

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CHAPTER 3: Mechanism of Substrate Hydroxylation 146 Substrate Dealkylation The increase in imino oxy carbon bond length for the copper alkoxide species (figure 3.11) was observed to be constant over the reaction coordinate until the energetic saddle point was reached. It was here that the substrat e bond length was dramatically increased as compared to the effect obs erved studying the cupric-hydroxyl species, ranging from 1.42 to 2.91 respectively. The corre sponding poses for the quantum mechanically treated region of the CuII OH simulation showed very little change in substrate orientation, with major functiona lities of the of the benzaldehyde imino-oxy acetic acid substrates (phenyl ring, imi no-oxy, and carboxy termini moieties) remaining constant with respect to starting points. At the saddle point, an increase in imino oxy carbon bond length was observed, a 1.44 change in an RC = 0.306 increment with the final bond length change 1.54 respectively. Discussion De-alkylation is Non-Enzymatic Oxidation of benzaldehyde iminooxy acetic acid via PAM yields the hypothesized benzaldoxime product, as determ ined by NMR (figure 3.1). The observed 13C chemical shift represents a benzaldoxime speci es. This is expecte d, as lifetimes of a benzaldehyde imino-oxy O -radical would be considerab ly short in an aqueous environment. This product was shown to be released from the enzyme with an equivalent and simultaneously formed amount of glyoxylate (figure 3.2). This result coupled with the observed ratio of [oxygen]-consumed : [benzaldoxime]-produced showing a 1:1 stoichiometry give strong evidence for the tight coupling observed for D M and PHM using natural substrates (figures 3.3 and table 3.1 )[3, 90]. From the de-alkylation study,

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CHAPTER 3: Mechanism of Substrate Hydroxylation 147 the rates of the PAL catalyzed -hydroxylN -benzoylglycine benzamide reaction were observed to occur unperturbed in the presence of varying concentra tions of benzaldehyde imino-oxy acetic acid. Conversely, competing -hydroxylN -benzoylglycine against the benzaldehyde imino-oxy acetic acid benzaldoxime reaction also displayed independent product formation, respectively (figures 3.5 and 3.6). This is strong evidence that the de-a lkylation of both substrates fo rmed product independent of each other. Consequently, it suggests that th e oxidative de-alkylat ion of benzaldehyde imino oxy acetic acid to benzaldoxime and glyoxyl ate require only the PHM domain of bi-functional PAM and neither r eactants nor products of this reaction interfere with the PAL-dependent de-alkylation of -hydroxylN -benzoylglycine. Kinetic Mechanism The minimal mechanism determined from primary deuterium kinetic isotope effects show a very unique trend (figures 3.7-3.8). Previous work with N -benzoylglycine[27] and N -acetylglycine (refer to CHAPTER 2) show the deuterium kinetic isotope effect for C H bond cleavage displaying D(VMAX/KM) values for both substrates to be equal and constant as tensi ons of the second substrate was altered. This trend suggested that the kinetic order fo r substrate addition to PHM was oxidizable substrate then oxygen, with an equilibrium-ordered minimal kinetic mechanism[91-93]. Comparison of equation 3.4 and 3.5, show that KM for the first bound substrate (KM,A[B] term) is absent from the denominator of the equilibrium preferred mechanism. In this case, since binding of the first substrate to the free enzyme (E) is in equilibrium, the dissociation constant (KIA[B] term) is all that is require d for this minimal mechanism, with respect to the first bound substrate. Evidence for steady-state minimal reaction

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CHAPTER 3: Mechanism of Substrate Hydroxylation 148 mechanisms with respect to PHM, was observed when the residue tyrosine 318 is mutated to phenylalanine, resp ectively [63]. Analysis of deuterium kinetic isotope effects for a steady-state preferred minimal mechanism, will include KM for the first bound substrate (KM,A[B] term) in the denominator, making this a more symmetrical mechanism comparatively. Therefore, the D(VMAX/KM) kinetic isotope effect observed for C H/D bond cleavage will (unl ike the equilibrium-preferred mechanism) be dependent on the concentration of the second substrate, resp ectively. The difference in a steady-state ordered versus random minima l mechanisms determined through kinetic isotope effects display D(VMAX/KM) values which approach unity as concentration of the second substrate becomes infinite for the orde red versus non-unity limits for the random case. The point mutation of tyrosine 318 to phenylalanine in PHM display D(VMAX/KM)A (where A is N -benzoylglycine, respectivel y) values of 8.32 2.32 mM-1s-1 as the concentration of oxygen approach es infinity[63]. In D M, the absence of the anionic activator (fumarate) at pH 6.0 alters the steady-state minimal mechanism from ordered to random [73, 94]. Kinetic isotope effect analysis of benzaldehyde imino-oxy acetic acid versus oxygen concentration show a dramatic decrease in the magnitude of D(VMAX/KM) for substrate C H/D cleavage as a function of oxygen tension. Replots (figures 3.7 and 3.8) display D(VMAX/KM) for benzaldehyde imino-oxy acetic acid which range from well beyond semi-classical values as [O2] 0 M (KIE 14.7) decreasing to within unity as [O2] respectively. The D(VMAX/KM) for oxygen remained constant over the experimental range studied. This is a very clear example of a steady-state ordered

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CHAPTER 3: Mechanism of Substrate Hydroxylation 149 mechanism with benzaldehyde imino-oxy acetic acid adding to PHM prior to di-oxygen, respectively according to scheme 3.8. E E-AG E-O2-BIAA E-(OH)BIAA-H2O E Benzaldoxime + k 1BIAA[BIAA] k2BIAA k 3O2[O2] k4O2k5k7 Scheme 3.8. Minimal kinetic mechanism determined for benzaldehyde imino-oxy acetic acid oxidation as determined through primary deuterium kinetic isotope effects. The dissociation constant for Benzaldehyde imino oxy acetic acid showed a normal isotope effect of DKD,BIAA = 2.06 0.52 (table 3.4). Conversely, N benzoylglycine has a reported inverse binding isotope effect which has been observed in both PHM and PAM [27]. The effect of isotopic labeling on binding is usually dismissed as negligible, though several syst ems have exhibited this phenomenon. For example lactate dehydrogenase[95], hexokina se[96], thymidine phosphorylase[97], and purine nucleoside phosphorylase[98] all exhi bit binding isotope effects. The DKD,BIAA suggests that there is likely a stringent vibrational contri bution associated with ground state activation/de-s tabilization processes, such as geometric distortion, during benzaldehyde imino-oxy acetic acid binding to the free enzyme. This result becomes especially interesting for reactions governed by hydrogen tunneling, like PHM and D M[63, 64]. As discussed in the previous chapter, the distance between hydrogen donor and the Cu/O acceptor can directly aff ect the probability of degenerate wave overlap between each group and is sensit ive to isotopic labeling[64, 99, 100]. Furthermore, CHAPTER 2 demonstrated a structural dependence associated with degenerate wave overlap suggesting that geometric distortion of the substrate may significantly contribute to the electronic/vibrational requirement for efficient substrate pre-organization. Therefore, the presence of DKI,BIAA suggest that the PHM domain

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CHAPTER 3: Mechanism of Substrate Hydroxylation 150 distorts benzaldehyde imino-oxy acetic acid geometry and that this phenomenon is reduced in the presence of di-deuteration at the -carbon, respectively. The (VMAX/KM) kinetic isotope effect for be nzaldehyde imino-oxy acetic acid reduced with increasing oxygen concentration conditions due to a phenomenon referred to as a commitment[93, 101, 102]. This term, introduced by Northrop and Cleland, applies to the monooxygenase reactions of D M and PHM as the rate of C H bond cleavage (chemical step) to the dissociation of the reactant from the central complex (E-RC -H-O2). The dominant enzyme form at infinite oxygen concentration becomes the central complex. Under steady-state orde red conditions, as di -oxygen concentration approaches infinity, dissociation of the reactant (RC -H) from the central complex does not occur as the commitment to the forward reaction also becomes infinite. Conversely, the magnitude of the (VMAX/KM) kinetic isotope e ffect on benzaldehyde imino-oxy acetic acid as [O2] 0 M, has a commitment that is dependent on the ratio of the chemical step versus the dissociat ion of di-oxygen from the central complex (scheme 3.8). Therefore, the predominant enzyme form will be the E-RC -H ternary complex due to the diminishing micro-rate constant for di-oxygen insertion into the central complex, k3[O2]. Due to their faster binding rate constants, glycine-extended peptide substrates display minimal kinetic mechanisms that are not equilibrium-ordered and instead characterized as steady-state ordered[26, 63]. This observation, taken in context with current benzaldehyde imino-oxy acetic acid study suggest that the iminooxy moiety may facilitate a sl ightly altered binding motif, as compared to non-peptide glycine-extended substrates. The details of this difference involve the manner in which glycine-extended substrate dissociation occurs from the ternary complex, respectively.

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CHAPTER 3: Mechanism of Substrate Hydroxylation 151 Dissociation constants for benzaldehyde imi no-oxy acetic acid are much more favorable than the values observed for N -benzoylglycine w ith wild-type PHM[27, 63]. While both substrates show no appreciable kinetic isotope effect on VMAX these maximal rates display an approximate six-fold decrease wh en benzaldehyde imino-oxy acetic acid is used, respectively. This s uggests that product release is slow compared to C H cleavage in this mechanism, justifying a DVMAX value equal to unity or much less than kH/ kD values. D(VMAX/KM) measured versus oxygen tension is the second-order rate constant which measures all steps for oxygen asso ciated with di-oxygen binding up to and including the hydrogen abstract ion step[26]. The constant magnitude observed suggests that the di-oxygen ac tivation step for C H/D bond cleavage is dependent on the isotopic character of the donor species. The magnitudes of the D(VMAX/KM)OXYGEN are not observed to extend beyond a semi-classical ra nge for either substrate and shown to be within two KIE units of each other. The VMAX/KM (s-1M-1) for oxygen is observed to be ~3.5 fold lower for protiated benzaldehyde imino-oxy acetic acid compared with N -benzoylglycine[27]. For the results presented, this decrea se in product release (VMAX) for benzaldehyde imino-oxy acetic acid cannot be attributed to an uncoupling of oxygen consumption to benzaldox ime and glyoxylate formation ( see figure 3.2), nor can it be explained through C H/D substitution, as these are very similar to N benzoylglycine. Therefore, the only logical remaining step is that the imino-oxy moiety is complicating C O insertion into its respective C -radical species. This preliminary conclusion sets benzaldehyde imino-oxy acetic ac id de-alkylation in the minimal kinetic mechanism as able to include no othe r micro-rate constant, other than k7. This micro-

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CHAPTER 3: Mechanism of Substrate Hydroxylation 152 rate constant would be the slow step responsible for the irre versible conversion of the E-P complex to free enzyme (E) and product fr om the oxidative de-alkylation process. Substrate Oxidation and Product Release are Uncoupled Quantum mechanical/molecular mechan ical (QM/MM) reaction coordinate simulations of oxygen insert ion into the benzaldehyde imino-oxy acetic acid C radical using the experimentally determined de-alkylation process as a probe into the nature of the Cu/O oxidant species. Following hydroge n abstraction from the substrate donor, the Chen/Solomons mechanism postu late a direct pathway for substrate hydroxylation from the homolysis and subsequent radi cal recombination of the copperII-hydroperoxo intermediate (CuII-OOH) producing a cupric-oxyl radical (CuII O) and a hydroxyl radical which recombines with the substrate derived C radical to yield -hydroxylated product, respectively[23, 61]. The Crespo and Yoshizawa mechanisms for substrate hydroxylation each involve the stabilization of a highly reduced copper-oxyl species for hydrogen atom abstraction, followed by a concer ted rebinding step an alogous to the P450 reaction[57, 59, 60]. These thr ee mechanisms are similar with respect to the manner in which substrate derived C -radicals are functionalized. Each of these mechanisms directly transfers the hydroxyl moiety to product, prompting simultaneous release of -hydroxy product, respectively. This concep t can be considered a free-product generation, as the nature of the copper/oxyge n oxidant species woul d interact with the substrate C -radical for only one step resulting in the simultaneous substrate oxidation and product release, respectively. Similar to the Chen/Solomons mechanism, the Klinman mechanism also utilizes a copperII-superoxo species for its hydrogen

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CHAPTER 3: Mechanism of Substrate Hydroxylation 153 abstraction mechanism. Following this event, the release of the distal OH moiety of the copperII-hydroperoxo species is releas ed as water via an intramolecular electron transfer between the copper domains. The subsequent copperII-oxyl radical forms an inner-sphere alcohol or copper-alkoxide complex with the substrate radical. This mechanism adds a step to the product release event observed in the free-product mechanisms, through the uncoupling of substrate C -radical oxidation from its subsequent release as C -hydroxylated product. The experimental design to differentia te these modes of product release was probed computationally through QM/MM reac tion coordinate simulation using an experimentally determined de alkylation event for mode selection. The CuII OH (singlet) species was postulated to address the direct OH transfer and product release characteristics detailed in the Chen/Solom ons, Crespo, and Yos hiwaza mechanisms. The effect of direct product formation on the bond length of our de -alkylation probe was negligible, sugges ting that imino oxy C bond dissociation would likely not occur through this mechanism. Formation of the copper alkoxide species (quartet) had a dramatic effect on the structure of the de-a lkylation probe, suggestin g that the process for substrate oxidation and product release must be completely uncoupled steps, and likely pass through a copper-alkoxide intermed iate (red coordinates, figure 3.12).

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CHAPTER 3: Mechanism of Substrate Hydroxylation 154 Reaction Coordinate [(C -O)-(Cu-O)] -3-2-10123 Potential Energy (kcal/mol) -40 -20 0 20 40 60 80 NO C Bond Distance (Angstrom) 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 (Cu-OH) RC vs G (Cu-OH) RC vs NO-C Bond Dist. (Cu-Alkoxide) RC vs G (Cu-Alkoxide) RC vs NO-C Bond Dist. Figure 3.12. Combined plot of QM/MM reaction coordinate simulation comparing the Cu-OH (singlet) and the CuII-O (quartet) species again bond distance fo r the benzaldehyde imino-oxy acetic acid dealkylation event, respectively. Please note, squa res represent the distance change between the NO C bond distance while the circles signify Cu O bond distance changes over the reaction coordinate. For more detail, please see methods section. Conclusion Addressed in the introduction, this is an exciting time to study oxygen activation filled with well defined copper oxygen species. With Kar lin, Solomon, Cramer and Roth groups leading the fi eld through elegant metal oxygen model studies, precedence for proposed species and definition of their rich chemistry are available becoming an invaluable foundation to understand oxygen activ ation events as a function of protein ligand environments[1, 48, 58, 103-107]. From the data presented, the benzaldehyde imino-oxy acetic acid appears to be th e only known example of a non-peptide, nonglycine extended substrate for PAM to displa y a steady-state ordere d bi-reactant minimal mechanism. This mechanism has only been observed with glycine-extended

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CHAPTER 3: Mechanism of Substrate Hydroxylation 155 substrates[26]. The tight-coupli ng of all reactants and products is consistent with all glycine-extended substrate data collected, to date [90]. Ben zaldehyde imino-oxy acetic acid is an interesting substrate for PAM. It is mechanistically equivalent substrate to in vitro glycine-extended peptides while also performing as a probe of the monooxygenase domain (PHM) within bi-functional PAM. Design of the de-alkylation event based solely on the oxidation chemistry of PHM, allowed the nature of substrate oxidation to be studied. The strong mixture of both, in vitro and in silico experimentation has revealed that direct hydroxyl insertion into the C -radical of benzaldehyde imino-oxy acetic acid is unlikely. This mechanism would likely prompt a complete uncoupling of benzaldehyde imino-oxy acetic acid radical s ubstrate and a reduced oxygen species from the PHM active site, as the substrate radical would resist conversion into a thermodynamically unstable hemiacetal derivative. Formation of the inner-sphere alcohol complex during benzaldehyde iminooxy acetic acid oxidation is the only mechanism that can account for the tight c oupling while still displaying the ability to non enzymatically de-alkylate. Therefor e, production of an oxidized copperII alkoxide intermediate complex would best facilitate the tightly coupled reaction characteristics observed with respect to the de alkylation process. This result provides evidence that substrate oxidation in PHM occurs through a covalently linked copperII oxyl substrate complex. The O de alkylation observed for this reaction has been predicted according to scheme 3.9.

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CHAPTER 3: Mechanism of Substrate Hydroxylation 156CuM IIO N O H COOCuM IIO N O H COOH+CuM IIO N OH H COOO H H OH2 HisHis Met HisHis Met HisHis Met Radical Recombination for CuII-Alkoxide Intermediate Formation Solvent-Assisted O -De-Alkylation of CuII-Alkoxide Intermediate Release of Benzaldehyde and Glyoxylate Products Scheme 3.9. De-alkylation reacti on predicted for the CuII-alkoxide intermediate formation with benzaldehyde imino-oxy acetic acid as the substrate. Future Aspects to This Study Our proposal that the Cu-Ospecies is the likely oxidant is based on the dealkylation characteristics unique to benzal dehyde imino-oxy acetic acid. The proposed quartet spin state would be paramagnetic and therefore a detectable phenomenon by either EPR (X-band or high fiel d) or NMR techniques, to name a few. Detection of this intermediate along the reaction coordinate coupled with prev ious kinetic isotope effect studies would be a decisive re sult confirming present conclusions. With this intermediate defined, the exact window for intra-molecu lar electron transfer could be estimated, making the re-oxidation of half or all of the copper domains possible to be measured. With general EPR methods, such as X-band, th e intermediates present may be silent. Though, beyond high field EPR or NMR techniques to trap transients, a novel area well suited to elucidate the electron transfer event is protein liga tion. Here, inteins would be used to reconstruct the PHM active site with a radical trap substituted for a residue[108110].

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CHAPTER 3: Mechanism of Substrate Hydroxylation 160 48. Lee, D.H., et al., Copper(I) Complex O(2)-Reactiv ity with a N(3)S Thioether Ligand: a Copper-Dioxygen Adduct Including Sulfur Ligation, Ligand Oxygenation, and Comparisons with All Nitrogen Ligand Analogues. Inorg Chem, 2007. 49. Fujisawa, K., et al., A monomeric side-on superoxocopper(II) complex: Cu(O2)(HB(3-tBu-5-iPrpz)3). J Am Chem Soc, 1994. 116 : p. 12079-12080. 50. Chen, P., et al., Spectroscopic and electronic structure studies of the diamagnetic side-on CuII-superoxo complex Cu(O2) [HB(3-R-5-iPrpz)3] : antiferromagnetic coupling versus covale nt delocalization. J Am Chem Soc, 2003. 125 (2): p. 46674. 51. Karlin, K.D., et al., Kinetic, thermodynamic, and spectral characterization of the primary copper-oxygen (Cu-O2) adduct in a reversibly formed and structurally characterized peroxo-dicopper(II) complex. J Am Chem Soc, 1991. 113 : p. 58685870. 52. Karlin, K.D., et al., Kinetics and thermodynamics of formation of copper-oxygen adducts: oxygenation of mononuclear copper(I) complexes containing tripodal tetradentate ligands. J Am Chem Soc, 1993. 115 : p. 9506-9514. 53. Karlin, K.D., S. Kader li, and A.D. Zuberbuhler, Kinetics and thermodynamics of copper(I)/dioxygen interaction. Acc Chem Res, 1997. 30 : p. 139-147. 54. Schatz, M., et al., Dioxygen complexes: Combined spectroscopic and theoretical evidence for a persistent endon copper superoxo complex. Angew. Chem. Int. Ed., 2004. 43 : p. 4306-4363. 55. Chen, P. and E.I. Solomon, Frontier molecular orbital analysis of Cu(n)-O(2) reactivity. J Inorg Biochem, 2002. 88 (3-4): p. 368-74. 56. Maiti, D., et al., A 1:1 copper-dioxygen adduct is an end-on bound superoxo copper(II) complex which undergoes o xygenation reactions with phenols. J Am Chem Soc, 2007. 129 (2): p. 264-5. 57. Crespo, A., et al., The catalytic mechanism of pep tidylglycine alpha-hydroxylating monooxygenase investigated by computer simulation. J Am Chem Soc, 2006. 128 (39): p. 12817-28. 58. Roth, J.P., Advances in studying bioinorganic reaction mechanisms: isotopic probes of activated oxygen intermediates in metalloenzymes. Curr Opin Chem Biol, 2007. 59. Kamachi, T., et al., Computational exploration of the catalytic mechanism of dopamine beta-monooxygenase: modeling of its mononuclear copper active sites. Inorg Chem, 2005. 44 (12): p. 4226-36. 60. Yoshizawa, K., et al., Catalytic mechanism of dopamine beta-monooxygenase mediated by Cu(III)-oxo. Inorg Chem, 2006. 45 (7): p. 3034-41. 61. Chen, P., et al., Oxygen activation by the noncoupled binuclea r copper site in peptidylglycine alpha-hydroxylating monooxygenase. Spectroscopic definition of the resting sites and the putat ive CuIIM-OOH intermediate. Biochemistry, 2004. 43 (19): p. 5735-47. 62. Guallar, V. and R.A. Friesner, Cytochrome P450CAM enzy matic catalysis cycle: a quantum mechanics/molecu lar mechanics study. J Am Chem Soc, 2004. 126 (27): p. 8501-8.

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CHAPTER 3: Mechanism of Substrate Hydroxylation 161 63. Francisco, W.A., N.J. Bl ackburn, and J.P. Klinman, Oxygen and hydrogen isotope effects in an active site tyrosine to phen ylalanine mutant of peptidylglycine alphahydroxylating monooxygenase: mechanistic implications. Biochemistry, 2003. 42 (7): p. 1813-9. 64. Francisco, W.A., et al., Hydrogen tunneling in peptidylglycine alphahydroxylating monooxygenase. J Am Chem Soc, 2002. 124 (28): p. 8194-5. 65. Thomas, J.R., Electron Spin Resonance Study of Iminoxy Free Radicals. J. Amer. Chem. Soc., 1964. 86 (7): p. 1446-1447. 66. Miller, D.A., et al., Characterization of a bifunctional peptidylglycine alphaamidating enzyme expressed in Chinese hamster ovary cells. Arch Biochem Biophys, 1992. 298 (2): p. 380-8. 67. Liu, K.-C., B.R. Shelton, and R.K. Howe, A Particularly Convenient Preparation of Benzohydroximinoyl Chloride s (Nitrile Oxide Prescursors). Journal of Organic Chemistry, 1980. 45 (19): p. 3917-1919. 68. McIntyre, N.R., et al., Thiorphan, tiopronin, and relate d analogs as substrates and inhibitors of peptidylglycine alpha-amidating monooxygenase (PAM). FEBS Lett, 2006. 580 (2): p. 521-32. 69. Merkler, D.J., et al., 18O isotopic 13C NMR shift as proof that bifunctional peptidylglycine alpha-amidati ng enzyme is a monooxygenase. Biochemistry, 1992. 31 (32): p. 7282-8. 70. Buettner, G.R. and B.A. Jurkiewicz, Catalytic metals, ascorbate and free radicals: combinations to avoid. Radiat Res, 1996. 145 (5): p. 532-41. 71. Samuni, A., et al., On the cytotoxicity of vitamin C and metal ions. A site-specific Fenton mechanism. Eur J Biochem, 1983. 137 (1-2): p. 119-24. 72. Cleland, W.W., Statistical analysis of enzyme kinetic data. Methods Enzymol, 1979. 63 : p. 103-38. 73. Klinman, J.P., H. Humphries, and J.G. Voet, Deduction of kinetic mechanism in multisubstrate enzyme reactions from trit ium isotope effects. Application to dopamine beta-hydroxylase. J Biol Chem, 1980. 255 (24): p. 11648-51. 74. Miller, S.M. and J.P. Klinman, Deduction of kinetic mechanisms from primary hydrogen isotope effects: dopamine be ta-monooxygenase--a case history. Methods Enzymol, 1982. 87 : p. 711-32. 75. Prigge, S.T., et al., Amidation of bioactive pep tides: the structure of peptidylglycine alpha-hydroxylating monooxygenase. Science, 1997. 278 (5341): p. 1300-5. 76. GLIDE, Schrdinger, LLC 2000, Portland, OR. 77. QSITE, Schrdinger, LLC 2000, Portland, OR. 78. Friesner, R.A., et al., Glide: a new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. J Med Chem, 2004. 47 (7): p. 1739-49. 79. Gratteri, P., C. Bonaccini, and F. Melani, Searching for a reliable orientation of ligands in their binding s ite: comparison between a st ructure-based (Glide) and a ligand-based (FIGO) approach in the case study of PDE4 inhibitors. J Med Chem, 2005. 48 (5): p. 1657-65.

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CHAPTER 3: Mechanism of Substrate Hydroxylation 162 80. Altun, A., S. Shaik, and W. Thiel, Systematic QM/MM investigation of factors that affect the cytochr ome P450-catalyzed hydrogen abstraction of camphor. J Comput Chem, 2006. 27 (12): p. 1324-37. 81. Banks, J.L., et al., Integrated Modeling Program, Applied Chemical Theory (IMPACT). J Comput Chem, 2005. 26 (16): p. 1752-80. 82. Wirstam, M., S.J. Lippard, and R.A. Friesner, Reversible dioxygen binding to hemerythrin. J Am Chem Soc, 2003. 125 (13): p. 3980-7. 83. Becke, A.D., A new mixing of HartreeFock and local density-functional theories. J Chem Phys, 1993. 98 (2): p. 1372-1377. 84. Becke, A.D., Density-functional thermo chemistry. III. The role of exact exchange. J Chem Phys, 1993. 98 (7): p. 5648-5652 85. Hay, J.P. and W.R. Wadt Ab initio effective core potentials for molecular calculations. Potentials for K to Au including the outermost core orbitals. J. Chem. Phys., 1985. 82 (1): p. 299-310 86. Cui, Q., Elstner, M., Kaxiras, E., Frauenheim, T., and Karplus, M., A QM/MM Implementation of the Self-Consistent Charge Density Func tional Tight Binding (SCC-DFTB) Method. J. Phys. Chem. B, 2001. 105 (2): p. 569-585. 87. Jorgensen, W.L., Tirado-Rives, J., The OPLS Force Field for Proteins. Energy Minimizations for Crystals of Cyclic Peptides and Crambin. J. Am. Chem. Soc. 1988. 110 : p. 1657-1666. 88. Kaminski, G.A., Friesner, R.A., Tirado-Rives, J., Jorgensen, W.L., Evaluation and Reparametrization of the OPLS-AA For ce Field for Proteins via Comparison with Accurate Quantum Chemic al Calculations on Peptides. J. Phys. Chem. B 2001. 105 p. 6474-6487. 89. MAESTRO, Schrdinger, LLC 2002, Portland, OR. 90. Evans, J.P., N.J. Blackburn, and J.P. Klinman, The Catalytic Role of the Copper Ligand H172 of Peptidylglycine alpha-Hydr oxylating Monooxygenase: A Kinetic Study of the H172A Mutant. Biochemistry, 2006. 45 (51): p. 15419-15429. 91. Cook, P.F. and W.W. Cleland, Mechanistic deductions fr om isotope effects in multireactant enzyme mechanisms. Biochemistry, 1981. 20 (7): p. 1790-6. 92. Cleland, W.W., The use of isotope effects to determine enzyme mechanisms. Arch Biochem Biophys, 2005. 433 (1): p. 2-12. 93. Cleland, W.W., Use of isotope effects to elucidate enzyme mechanisms. CRC Crit Rev Biochem, 1982. 13(4): p. 385-428. 94. Miller, S.M. and J.P. Klinman, Secondary isotope effects and structure-reactivity correlations in the dopamine beta-monoo xygenase reaction: evidence for a chemical mechanism. Biochemistry, 1985. 24 (9): p. 2114-27. 95. LaReau, R., W. Wah, and V. Anderson, Isotope effects on binding of NAD+ to lactate dehydrogenase. Biochemistry, 1989. 34 : p. 6050-6058. 96. Lewis, B.E. and V.L. Schramm, Binding equilibrium isotope effects for glucose at the catalytic domain of human brain hexokinase. J. Am Chem Soc, 2003. 125 : p. 4785-4798. 97. Birck, M.R. and V.L. Schramm, Binding causes the remote [5'-3H]thymidine kinetic isotope effect in human thymidine phosphorylase. J. Am Chem Soc, 2004. 126 : p. 6882-6883.

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CHAPTER 3: Mechanism of Substrate Hydroxylation 163 98. Murkin, A.S., et al., Neighboring group participation in the transition state of human purine nucleoside phosphorylase Biochemistry, 2007. 46 : p. 5038-5049. 99. Nagel, Z.D. and J.P. Klinman, Tunneling and dynamics in enzymatic hydride transfer. Chem Rev, 2006. 106 (8): p. 3095-118. 100. Klinman, J.P., The role of tunneling in enzyme catalysis of C-H activation. Biochim Biophys Acta, 2006. 1757 (8): p. 981-7. 101. Northrop, D.B. and Y.K. Cho, Effects of high pressure on solvent isotope effects of yeast alcohol dehydrogenase. Biophys J, 2000. 79 (3): p. 1621-8. 102. Blanchard, J.S. and W.W. Cleland, Use of isotope effects to deduce the chemical mechanism of fumarase. Biochemistry, 1980. 19 (19): p. 4506-13. 103. Decker, A. and E.I. Solomon, Dioxygen activation by copper, heme and non-heme iron enzymes: comparison of elec tronic structures and reactivities. Curr Opin Chem Biol, 2005. 9 (2): p. 152-63. 104. Lanci, M.P., et al., Isotopic Probing of Molecular Oxyg en Activation at Copper(I) Sites. J Am Chem Soc, 2007. 129 (47): p. 14697-14709. 105. Cramer, C.J. and W.B. Tolman, Mononuclear Cu-O2 complexes: geometries, spectroscopic properties, electro nic structures, and reactivity. Acc Chem Res, 2007. 40 (7): p. 601-8. 106. Heppner, D.E., et al., Can an ancillary ligand lead to a thermodynamically stable end-on 1 : 1 Cu-O2 adduct supported by a beta-diketiminate ligand? Dalton Trans, 2006(40): p. 4773-82. 107. Aboelella, N.W., et al., Effects of thioether substituents on the O2 reactivity of beta-diketiminate-Cu(I) complexes: probing the role of the methionine ligand in copper monooxygenases. J Am Chem Soc, 2006. 128 (10): p. 3445-58. 108. Seyedsayamdost, M.R., C.S. Yee, and J. Stubbe, Site-specific in corporation of fluorotyrosines into the R2 subunit of E. coli ribonucleotide reductase by expressed protein ligation. Nat Protoc, 2007. 2 (5): p. 1225-35. 109. Seyedsayamdost, M.R., et al., pH Rate profiles of FnY356-R2s (n = 2, 3, 4) in Escherichia coli ribonucleoti de reductase: evidence t hat Y356 is a redox-active amino acid along the radi cal propagation pathway. J Am Chem Soc, 2006. 128 (5): p. 1562-8. 110. Yee, C.S., et al., Generation of the R2 subunit of ribonucleotide reductase by intein chemistry: insertion of 3-nitrotyr osine at residue 356 as a probe of the radical initiation process. Biochemistry, 2003. 42 (49): p. 14541-52.

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid Introduction The amidation of neuropeptide hormones is a ubiquitous post-translational modification catalyzed by peptidylglycine -amidating monooxygenase (PAM) and is the rate-limiting step for bio-activation of glycine-extended pro-hormones ( 1, 2). The in vivo localization of PAM is not sole ly restricted to neuroendocri ne tissues, with high amounts also found in atrial myocytes(3 ). Other sites for the tissu e localization of PAM include bronchial cartilage, smooth muscle cells, airway and olfactory epithelium(4), as well as endothelial cells(1, 5). The oxidation chemistry for members of the non-coupled binuclear copper monooxygenase family, PA M and its sister enzyme dopamine monooxygenase(D M), involve the prereduction of enzyme-bound coppers followed by the sequential addition of both oxygen and oxi dizable substrate to form the central complex( 6). The nucleophilic oxygen is activated through coordination to the reduced active site copper, referred to as CuM (two histidines and one m ethionine). This copperIIsuperoxo species accepts the methylene hydrogen from the docked substrate followed by di-oxygen cleavage of the copperII-hydroperoxo to yield water and hydroxylatedmethylene (PROH) products, respective ly (scheme 4.1). 164

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid S RHO2H2O EESRHESRHO2EPROHE PROH Scheme 4.1. Kinetic order for PAM and D M oxidation reactions. SRH and PROH represent the unactivated al kyl substrate and oxidized product. E, ES, ESRHO2, and EPROH represent enzyme states present during the reaction coordinate from free enzyme (E), ternary complexes (ESRH and EPROH), and the central complex (ESRHO2). The reaction mechanism for both PAM and D M appear super imposable, though differences occur with the endogenous substrates functionalized by each. In vivo PAM oxidizes glycine-terminal peptide-prohormones( 7) and (likely) corresponding fattyacids(8, 9 ) while D M appears only to catalyze th e oxidation of dopamine to norepinephrine( 6, 10 ). Consistent with other meta llo-monooxygenases, these enzymes broad in vitro substrate specificity has been observed. From the heme-dependant cytochrome P450 enzymes (11) through coupled and non-coupled bi-nuclear copper enzymes tyrosinase/catechol oxidase( 12) and D M/PAM( 13-18), each metallomonooxygenase appears to have the ability to functionalize non-natural substrates. The oxidation of benzaldehyde imino-oxy acetic acid by PAM displaye d several unique reaction characteristics which warranted further study. The minimal kinetic mechanism showed a sequential C H bond activation that was dependent on the concentration of oxygen. This was an intriguing trend whic h caused the rate-lim itation of C-H bond cleavage at low oxygen to be very large decreasing to unity as oxygen concentration approached infinity. In add ition, the product release step (benzaldoxime) was observed to 165

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid result solely from a non-enzymatic O -dealkylation reaction that followed the monooxygenase reaction of PAM. Therapeutic Targeting of PAM Highly specific inhibition of PAM appears to be a viable therapeutic target because the overproduction of amidated pe ptides has been observed in a number of disease states, such as adrenomedulin in androgen-independent prostate cancer (19). Furthermore, amidated peptides such as s ubstance P and calcitonin gene-related peptide (CGRP) are also well-known moderators of inflammation( 20, 21). As a result, PAM has been implicated in chronic inflammation diseases such as Crohns disease and rheumatoid arthritis ( 20 ). Unfortunately, the targeting of PAM has been wrought by the absence of a highly specific inhibitor (22-24) or delivery problems associated with compounds which show nanomolar in vitro inhibition ( 25). For example, in vivo specificity of highly potent in vitro PAM inhibitors has been decreased as these compounds coordinate to the enzyme -bound metals of other proteins( 22). The sulfurmoiety of captopril displayed this trend, inhibiting angiotensi n-converting enzyme (ACE) as well as PAM through zinc (ACE) or copper (PAM) interaction(22). A similar result was observed in another study which pred icted thiolate chelation of the active-site CuH (copper domain composed of three histidin e residues) for tiopronin and thiorphan as the inhibition mechanism for PAM( 23). Unfortunately, th e PAM inhibition was nonspecific, as thiorphan also inhibits the metallo-enzyme neutral endopeptidase, and tiopronin is prescribed as a metal chelator for both copper and mercury poisoning( 23 ). A different mechanism for highly efficient PA M targeting has been the development of mechanism based inhibitors( 5, 20, 26, 27 ). These compounds are substrate analogues 166

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid which irreversibly inactivate an enzymatic r eaction as a consequence of turnover, leading to their common name as suicide inhibitors( 28-31). To date, 4-phe nyl-3-butenoic acid (PBA) has been the most extensively studi ed mechanism based inhibitor of PAM ( 32, 33). PBA is not an exclusively irreversible, mechanism-based inhibitor; as its efficiency is measured through a partition ratio which describes the specificity for inactivation compared with turnover or inhibitio n. PBA behaves as either a substrate or inactivator for PAM, turning over 100 times per one irrevers ibly inactivated PAM molecule ( 32 ). Currently, only oxidation metabo lites are known and the actual mechanism for PBA-dependant inactivation ha s not been experimentally determined (figure 4 .1). PBA is a very promising inactiv ator, though for rationa le design of nextgeneration mechanistic-based enzyme inhibito rs of PAM based on this scaffold, it is imperative that key points of the enzyme reaction are well-understood. Such as, definition of the rate determining step(s) ( 34 ), nature of radical intermediates/role of the olefin( 35), alteration in the natural kinetic order of substrate addition, and binding motifs as the transition state for the reaction is reached. These are necessary characteristics required to quantify tissue-, speciesand disease-sp ecific mechanism-based inactivation between PAM targets for design of the most selective therapeutic( 36). 167

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid O OH O OH O OH OH OH MECHANISM BASED INACTIVATOR? PAMPBA 2-OH-PBA 4-OH-PBA Figure 4.1. Summarized from Driscoll et al ( 32 ) displaying the oxidative metabolites of from 4-phenyl-3-butenoic acid (PBA) oxidati on. 2and 4-hydroxy derivatives of PBA were observed, though the inactivati on species is cu rrently unknown. Theoretical Tools for Structure-Function Analysis To de-convolute the unique chemistry observed for the imino-oxy acetic acids a structure-function analysis was used to explore components of the oxime (C=N O) moiety to determine their relationship with amide (C(O) NH ) counterparts observed in natural, glycine-extended substrates. To support this analysis, an in silico treatment of these compounds was also carried out to observe electron ic similarities of these structures in their pr edicted binding orientation w ithin PHM. The following sections will serve as an introduction to th eory and terminology util ized for this study. Molecular Electrostatic Potential versus Na tural Bond Orbital Population Analysis Molecular electrostatic potential (MEP) is an example of population analysis based on electrostatic potential MEP describes electronic dist ribution within a molecule by calculating the differences in interaction energy between the nucle i to describe the 168

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid potential energy surface (37, 38). The electrostatic potenti al depends directly on the wave function and, therefore, converges as the size of the basis set and amount of electron correlation is increased. MEP can be utilized for surface visualization providing information about local polarity trends using a color coding convention, with the most negative potential designated red and the most positive, blue. Therefore, these van der Waals surfaces can be separate d into regions of potential a llowing trends in reactivity to be analyzed. Natural bond orbital (NBO) analysis di ffers from MEP as it is a population analysis based on electron density ( 39). NBO originated as a technique for studying hybridization and covalency effect s in polyatomic wave functions( 40). It was implemented to obtain a deeper insight in to the electronic structure of optimized geometries. NBO analysis is based on a one-electron density matrix which defines atomic orbital shape within its molecula r environment, while molecular bonds are determined from the electron density between atoms. NBO analysis provides an orbital picture where wavefunctions closely corres pond to localized bonds and lone pairs in molecular structure. NBO analysis transforms the delocalized many-electron wave function into optimized electron-pair bonding units, corresponding to the Lewis structure picture (41). The set of high-occupancy Lewis-type (bond, lone pair) NBOs, each taken to be doubly occupied (two electrons), repres ent the natural Lewis structure of the molecule. Delocalization effects appear as w eak departures from this calculated structure as nonzero occupancies of non-Lewis NBOs. Therefore, non-Lewis occupancy becomes a quantitative measure of el ectronic delocalization. 169

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid Hyperconjugation Although hybridization is a fundamental concept of electronic and molecular structure, without high-symmetry molecular c onstraints, e.g. equivalent hybrid orbitals, there lacks a formal basis to this chemical theory( 42). For example, the prototypical tetrahedron formed by an sp3 hybrid orbital has four ma tching sigma bonds which detail equivalent bonds in the molecule. From the NBO localization method, hybridization can be assigned to both atomic l one pairs and to each atoms bonding orbitals, respectively. With NBO analysis, the percen t sand p-orbital character in a hybridized orbital is immediately evident from the coefficients of the atomic orbital (AO) from which the NBO is formed. In addition, population analys is can be carried out using the NBOs to derive atomic charges ( natural population analysis following sub-section). This introduces a critical concept referred to as hyperconjugation which rationalizes the chemical phenomena of filled-orbital empty-orbital interactions. This interaction describes the delocalization of negative charge, whereby the electrons in the filled orbitals are able to move partially into an unoccupied orbital. NBO analysis is well suited to quantify this phenomenon since th e NBOs are not determined by diagonalizing a Fock (or Kohn-Sham) operator( 43, 44). The Fock matrix of the NBO basis will contain non-zero off-diagonal elements (eigenvector s). Second order perturbation theory indicates that the values for these off-dia gonal elements between filled and empty NBOs can be interpreted as stabilizat ion energies originating from hyperconjugative interaction. 170

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid Natural Population versus Mulliken Analysis Mulliken populations( 45, 46 ) fail to give a useful and reliable characterization of the charge distribution in many cases and are sensitive to changes in basis set size and level of theory used( 39 ). Conversely, populations dete rmined with natural population analysis (NPA) are intrinsic to the wavefunc tion, rather than to a particular choice of basis orbitals, and are found to converge smoothl y toward well defined limits as the wave function is improved, with stable populations( 47). Contrasting Mulliken populations, natural populations exhibit constant values independent of basis se t choice. A striking similarity between Natural Population and Mulliken analysis methods describes the occupation of individual orbitals rather than of spatial regi ons. It should be illustrated that NPA orbital occupancies refer to the intrinsic natural atomic orbitals rather than to basis atomic orbitals (Mulliken), thus facilitating orbi tal-by-orbital comparison of wave functions calculated with different basis sets( 40 ). Therefore, natural population analysis represents the total electronic repulsions at a specific atom. This can be used to observe trends in molecular charge distribution 1 Second Order Perturbation Theory The second order interaction energy ( E2 ij) represents non-covalent hyperconjugation effects associated with donor and acceptor orbitals( 40). The interaction energy originates from the influe nce an occupied Lewis orbital structure has on an unfilled, anti-bonding orbital. The perturbative Fock matrix elements are related to 1 NPA is generally represented by the natural atomic ch arge of an atom. This is the difference of nuclear charge (Z) from the total electron population on that atom. The sum of natural charges over all atoms gives the net charge of the system. This is zero for neutral compounds and increases or decreases with the overall charge on a molecule. 171

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid the shapes of the bonding and anti-bonding orb itals through the NBO counterpart of the Mulliken approximation, NPA. This allows the noncovalent energy lowering to be described in terms of a generalized princ iple of maximum overlap between bonds and anti-bonds as defined by Bent ( 48). Bents Rule Hybridization in the cl assical sense, involves spn, where n equals an integer 1 though 3, respectively. The spn notation is shorthand for the %s versus the %p character of the hybrid. For example, sp3 has 25%s and 75%p character or s1/4p3/4. Bent formulated that hybridization could be enri ched to accommodate the electronegativity of inequivalent bonding ligands( 48). Thus, NBO analysis utilizes Bents rule to define n as any value between 0 and ( 42). This allows a working framework in which the mistaken notion that n must always equal 1, 2, or 3 can be shed, al lowing a more applicable and quantitative definition of hybridization to be defined. Precedence for NBO Analysis In an insightful review, Weinhold discu sses an elegant exampl e of NBO analysis to determine the origins for the internal rotation barrier of ethane( 44). Most organic texts describe the rotation barrier of ethane or iginating either from the steric repulsion of methyl hydrogens( 49) or as a function of pz-orbital overlap using molecular orbital (MO) theory( 50 ). The energy barriers displayed in figure 4.2, show that the staggered conformation to be the most stable equilibr ium geometry of ethane. Previous chemical reasoning for rotamer selection has been calle d into question as st eric repulsions have 172

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid been challenged as the sole determinant in ethane rotamer selection ( 51-54). These studies postulated that ethane requires hyperconjugation to fully explain the preference of the staggered rotamer. Hb Hb Hb Hb Hb Hb Hb Hb C Ha Ha Ha C Ha Ha Ha HaC Ha Ha HbC Hb Hb HaC Ha Ha Hb Hb HbCHaC Ha Ha HaC Ha Ha HbC Hb Hb E = 2.8 kcal mol-1Potential Energy (kcal mol-1)Dihedral Rotation Angle 06 01 2 0 Hb Ha Ha Hb Ha Hb Steric RepulsionHyperconjugative Stabilizationpzpz Figure 4.2. Potential energy barrier for the C C bond rotation within ethane. The selection of the thermodynamically-favored rotamer ( staggered ) is specified with respect to both steric repulsion and hypercon jugative stabilization models ( top ). Refer to text for more detail. 173

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid Hyperconjugative interactions between filled and unfilled orbitals define the energy barrier for rotation. Previous consideration was never given to the C H -bond of the methyl groups, as they were assumed to be degenerate( 50 ). Interestingly, NBO analysis showed strong CH delocalization into the adjacent CH* orbital resulting in increased hyperconjugative stab ilization in the staggered co nformation creating a partial double bond character referred to as vicinal hyperconjugative stabilization. Work by Goodman predicted hyperconjug ative effects to dominate the selection of ethane conformers( 52-54) instead of steric repulsive forces ( 51 ). This was an extremely controversial and counterintuitive proposal within the molecular structure community, making further calculations on the rotation barrie rs of ethane necessary. Recently, Mo and Goa calculate that, as predicted, ster ic repulsion predominan tly decides rotamer geometry, with moderate contributions fr om hyperconjugative stabilization (~30%)( 55). Therefore, the contribution of NBO to the analys is of ethane geometry is important as it validates the concept of an antiperiplanar delocalization between CH CH* orbitals. This is a significant contribution to the steric repulsion theory because it adds greater electronic detail to molecular structure pr eferences that determ ine the equilibrium geometry in ethane (figure 4.2). Structure-Function of the Imino-Oxy Acetic Acid The hypothesis of this work was that all imino-oxy acetic acids derivatives of glycine-extended substrate counterparts w ould be PAM substrates because the oxime moiety is electronically anal ogous to an amide. Buildi ng on the work introduced with the benzaldehyde imino-oxy acetic acid, a vari ety of para-substituted and naphthalene 174

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid containing acetophenone and benzaldehyde imino-oxy acetic acid derivatives were synthesized and tested for PA M activity. The st ructural dependence was further probed using primary kinetic isotope effects w ith two of these compounds (acetophenone and naphthaldehyde imino-oxy acetic acid) for direct comparison of rate-limiting chemistry with benzaldehyde imino-oxy acetic acid. This study also sought to define a structure-activity re lationship to describe the role imine-linked oxygen assumes in PA M-dependent oxidation of imino-oxy acetic acid substrate. As benzaldehyde imino-oxy ace tic acid is a constitutional isomer of N benzoylglycine and both are PAM substrates the role of the imine-linked oxygen was compared through oxygen insertion into glycine-containing molecules, making hydroxamic acid and hydroxylamine derivatives. Comparison of the kinetic behavior within this panel of compounds allowed th e effect oxygen insertion had on substrate oxidation to be experiment ally probed (Fig. 4.3). 175

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid H2N O OH glycine H2N O OH carboxymethylhydroxylamine O N O OH N -benzoylglycine O H NO OH O -acetylbenzohydroxamic acid O H O N O OH phenylacetylamino-acetic acid O H NO OH O H O phenylacetylamino-oxyacetic acid N O OH H O OH O N (benzylidene-amino)-acetic acid benzaldehyde imino-oxy acetic acid Figure 4.3 Structures of interest for the structur e-function analysis used in this study. 176

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid A multi-faceted approach using the comput ational docking of these compounds into PHM was utilized for structur e-function analysis. Structur es obtained from this method were used to rationalize the reactivity of the imino-oxy acetic acid moiety through MEP and NBO analysis. This coupled approach was performed to obtain a deeper insight into the electronic structur e of PHM-docked compounds to a fford a better understanding of the chemical reactivity observed w ith imino-oxy acetic acid substrates( 40, 47 ). With this approach, NBO analysis was used to rationali ze the role hyperconjugative stabilization contributes to observed trends for substrate functionalization (Fig. 4.4). 177

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid Computational Compound Docking Compound of Interest Kinetic Analysis Substrate KM, VMAXInhibitor KI Figure 4.4. Outline of structure-function analysis used for compounds used in this study. Single Point Enegy Calculation of Docked Geometry Electron Structure Analysis (MEP, NPA, NBO Analysis) Predicted Delocatization Pattern as a function of 'oxygen insertion' Correlation of Structure-Activity with Electronic Calculations to Rationalize Reactivity of the Imino-Oxy Acetic Acid 178

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid Materials and Methods All aryl aldehyde and ketone starting mate rials were purchased from Sigma Aldrich, O acetylbenzohydroxamic acid and phenylacetylamino-oxy acetic acid from Spec, dideutero bromoacetic acid from Sigma, carboxymethylhydroxylamine from TCI America, and all solvents and buffers were obtained from Fischer. Bovine liver catalase was purchased from Worthi ngton, bi-functional PAM (rattus novegus) was a generous gift from Unigene, Inc (New Jersey, USA), Synthesis of aryl imino-oxy acetic acids Synthesis of para-substituted aryl iminooxy acetic acids followed the procedures of van Dijk and Zwagemakers ( 56). Briefly, 80mmole of the respective aldehyde or ketone and carboxymethylhydroxylamine-hemichlori de were refluxed w ith 3 equivalents of NaOAc in 80% ethanol. The resulti ng solution was concen trated under reduced pressure, made alkaline with dilute sodium hydroxide (~100mM), then extracted with diethyl ether to remove the un-reacted ketone or aldehyde starting material. The aqueous phase was combined with fresh diethyl ether in a separatory funnel and was acidulated with a dilute hydrochloric aci d solution. The precipitant form ed in the aqueous phase was dissolved into the organic phase with agitation. Once precipitation was no longer observed, the organic phases we re separated. The combined organic layers were washed with brine, drie d (anhydrous MgSO4), and concentrated. Recrystallization was performed using a benzene-petroleum ether solution. -Dideutero Acetophenoneand -Naphthaldehyde imino-oxy acetic acid A method was for imino-oxy acetic acid s ynthesis was employed for the isotopic labeling studies. The ketone or aldehyde was converted to the corresponding oxime (57). The 4.1mmole oxime was dissolved in 20ml of ddH2O and 5 equivalents of NaOH. The 179

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid reaction was stirred at room temperature for 45 minutes, after whic h 1.5 equivalents of bromo-acetic acid was slowly added in sma ll increments to the stirring solution of sodium oximate. Upon completion the reac tion as determined by TLC (1:3 Hexanes : EtOAc), the reaction was acidified with dilute HCl(aq), collected in a Bchner funnel passing petroleum ether over the filtered solid to remove solvent from the filtrate. The white crystalline imino-oxy acetic acid solid was re-crystal lized twice with benzenepetroleum ether. para-(5-Dimethylaminonapthalene-1-sulfonamid o)acetophenone iminooxy acetic acid Distilled pyridine stored over molecula r sieves and potassium hydroxide pellets, was prepared for this procedure. Similar to the pyridine method used by Himel et al. ( 58), a 15mL aliquot of dry pyridine was pl aced in a closed round-bottom flask with a light N2 stream exchanging above the stirring so lution for ~15 minutes, while heating the solution to ~50-60 C. To this solution, 4 mmole of the para-amino acetophenone iminooxy acetic acid (synthesized from the aforementioned synthetic protocol) was dissolved in minimum of dry pyridine and added to th e stirring solvent thr ough the rubber septum with a syringe, followed by a short N2 exchange (~2 minutes). An equivalent amount dansyl chloride was dissolve d in 2 mL of dry pyridine, capped with a rubber septum allowing its headspace to be purged with N2. This solution was added drop wise to the stirring dry pyridine and para-amino acetophenone imino-oxy acetic acid solution. The sealed mixture was stirred for 24 hours. The reaction was monitored by TLC (20% methanol/ 80% ethyl acetate) wi th the product being resolved as bright fluorescent green spot. The reaction was diluted with ddH2O (~20 mL) and extracted with diethyl ether three times. Pyridine contamination of th e product was removed by rigorously stirring 180

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid the combined di-ethyl ether extracts in the presence of 2% hydrochloric acid(aq) solutions. This acidic solution was exchanged until TLC showed no pyridine contamination in either phase. The diethyl ether phase was se parated and concentrate d. Recrystallization was performed using hot diethyl ether usi ng petroleum ether to precipitate the yellow para-(5-dimethylaminonapthalene-1 -sulfonamido) acetophenone imino-oxy acetic acid product. The double recrystallized produc t was washed with petroleum ether in a Bchner funnel and dried in an oven prior to characterization. Steady State Kinetics using Oximetry Substrate Analysis Initial rates were measured by followi ng the PAM-dependent consumption of O2 using a Yellow Springs Instrument Model 53 oxygen monitor interfaced with a personal computer using a Dataq Instruments analogue /digital converter (model DI-154RS) as previously described in McIntyre et al. ( 23). Rate of dissol ved oxygen disappearance was used to determine steady-state values using equation 4.1, as fit to Kaleidagraph. rate VMAX,appB IAA@AKM IAA@A f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f Equation 4.1. Michaelis-Menton rate equation for initial velocity determination. Equation 4.1 describes the Michaelis-Menton expression, with initial velocity represented by rate, maximal velocity by VMAX, Michaelis constant by KM, and iminooxy acetic acid concentration by [IAA], respect ively. Reactions were performed under ambient oxygen conditions at 37.0 0.1 C (217 M)( 59). Each trial was buffered with 100mM MES/NaOH (pH 6.0), supplemented with 30mM NaCl, 1% (v/v)EtOH, 0.001% Triton X-100, 10 g/ml bovine liver catalase, 1 M Cu(NO3)2 and 5mM sodium ascorbate. In the presence of varying im ino-oxy acetic acid (IAA) subs trate, a background rate of 181

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid oxygen consumption was established and subtracted from the experime ntal rate initiated with PAM (82nM). Values for VMAX, app were normalized to published values for 11.0mM N -acetylglycine( 9). N-Acetylglycine Oxidation Analysis of hydroxamic acid derivative inhibition of PAM-dependant substrate oxidation was also carried out using the oxygen electrode, described above. Reaction conditions were repeated as above, adding a constant 5mM N -acetylglycine concentration to each trial. In the presence of varying inhibitor concentrations, initial rates were measured by following the decrease in PAM-dependent consumption of O2 using the previously mentioned oxygen mon itor apparatus. Values for VMAX, app were normalized to published values for 11.0mM N -acetylglycine( 9 ). The decreasing velocities for substrate oxidation fit to a competitive inhibition pattern described by Dixon plot for (equati on 4.2) using the Kaleidagraph graphing application. rate VMAX,APPB acetylglycineBC ffffffffffffffffffffffffffffffffffffffffffffffffffffffffff KM1 I@AKIffffffffg acetylglycineBC f f f Equation 4.2. Modified Michaelis-Menton expression to describe competitive inhibition. For competitive inhibition described thr ough Dixon analysis, rate is the initial velocity, VMAX is the maximal velocity, [acetylglycine] and [I] are N -acetylglycine and inhibitor concentrations and KM and Ki are the Michealis and inhibition constants, respectively. 182

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid Inhibition of N-dansyl-tyr-val-gly amidati on by imino-oxy acetic acid derivatives. Reactions at 37.0 0.1 C were initiated by the addition of PAM (45-50 ng) into 500 mL of 100 mM MES/NaOH pH 6.0, 30 mM NaCl, 5.0 mM sodium ascorbate, 1.0% (v/v) ethanol, 0.001% (v/v) Triton X-100, 10 mg/mL bovine catalase, 1.0 M Cu(NO3)2, 8 M N -dansyl-Try-Val-Gly, and various concentrations of the im ino-oxy acetic acid derivative. At the desired ti me, an aliquot was removed and added to a vial containing one-fifth volume of 6% (v/v) tr i-fluoroacetic acid to terminat e the reaction. The acidified aliquots were assayed for the percent conversion to N -dansyl-Try-Val-NH2 by reverse phase HPLC as described by Jones et al ( 60 ). Utilizing the fluorometric assay of Jones and Tamburini et al. ( 60), the amidation of the dansylated glycin e-extended tri-peptide ( N -dansyl-tyrosine-valine-glycine) was monitored via reverse-phase high performan ce liquid chromatography using an Agilent 1100. A constant fluorescen t-substrate concentration was measured against varying amounts of imino-oxy acetic acid, respectively. The decreasing velocities for fluorescent-substrate amidation were subseque ntly fit to a Dixon plot for competitive inhibition (equation 4.3) using the Ka leidagraph graphi ng application. 183

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid rate @AKM1 I@AKIffffffffg DNS @ YVG@A VMAX,APPB DNS @ YVG f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f Equation 4.3. Modified Michaelis-Menton expression to describe competitive inhibition. Please note, v is the initial velocity, VMAX is the maximal velocity, [DNS-YVG] and [I] are N -dansyl-tyrosine-valine-glyc ine and inhibitor (imino-oxy ac etic acid) c oncentrations and KM and KI are the Michaelis and inhibition constants, respectively. Reactions were performed at 37.0 0.1 C, 100mM MES/NaOH (pH 6.0), 30mM NaCl, 1% (v/v)EtOH, 0.001% Triton X-100, 10ug/ml bovine liver catalase, 1 M Cu(NO3)2, 8 M N -DansylYVG and 5mM sodium ascorbate. Duplic ate time points were collected through the removal 40ul of the reaction and subsequent mixture with 1% TFA with plotted data represented as initial velocity (s-1) standard deviation ve rsus corresponding [I]. Computation Similar to the analysis in CHAPTER TWO, the initial coordinates of the reduced PHM structure were utilized from the Protein Data Bank ( http://www.rcsb.org/pdb/ 1SDW)( 61). High accuracy subs trate binding poses were predicted using quantum polarized ligand docking ( QPLD) utilizing the molecular mechanics (MM) and ab initio programs of the Schrdinger Fi rst Discovery suites, Glide(62) and Q-site( 63) respectively( 64, 65). The top five Glide poses were selected using standard precision (SP) mode. These ligand-recep tor complexes are analyzed by ab initio methods to calculate partial atomic charges utilizing electr ostatic potential fitting within the receptor using the Q-site module. Specifically, a single-point energy op timization using the 6184

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid 31G* basis set and DFT-B3LYP( 66, 67 ) methodology was used to calculate the ligand wave-function deriving partial atomic char ges from molecular electrostatic potential (MEP) population analysis (68). These optimized ligands are re-docked to predict the lowest energy conformation of the ligand set. Electronic structure calculations Using the docked geometry calculated from the QPLD algorithm, compounds underwent an additional single-point energy ( SPE) optimization designed to predict both molecular electrostatic potential surfaces (MEP) and natural bonding orbital (NBO) analysis. Density functional theory (DFT ) calculations were performed using the Jaguar(69 ). Each calculation was generated with the 6-31G** basis set at the B3LYP level of theory. Surfaces were visualized in Maestro(70). Supplementary Note The structure of (benzylidine-amino)-acetic acid has a known sensitivity to moisture and air, decomposing to benzaldehyde ( 71). This degradation precluded kinetic analysis of this compound which was only included for theoretical comparison to benzaldehyde imino-oxy acetic acid to assist in understan ding the relationship be tween the imine and inserted oxygen. Results Steady State Kinetics of IminoOxy Acetic Acid Derivatives Steady-state analysis of all synthesized imino-oxy acetic acid derivatives show modest deviation in (VMAX/KM)APPARENT parameters, based on para-substitution (structures 1-15). The O -acetyl aryl-aldoxime and ketoxime derivatives show a 185

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid (VMAX/KM),APP ( s-1M-1) range of 1.51*104 to 1.31*105 and 2.11*104 to 1.12*105, respectively. The para-cyano benzaldehyde and the non-substituted acetophenone derivatives displayed the lowest (VMAX/KM)APPARENT values, while the greatest value was also found on different aldoxime (-OMe) and ket oxime (-Cl) para-substituted derivatives. The greatest (VMAX/KM)APPARENT value was observed for N -dansylated derivative 17, with a value of 4.11*105 s-1M-1. Overall, the KM,APPARENT values for the acetophenone iminooxy acetic acid derivatives were observed to be lower than th eir corresponding benzaldehyde counterparts, with the exception of para-methoxy ( 4 ten-fold lower than 11) and para-fluoro ( 6 13). Among the aryl structures 1-15, the VMAX,APPARENT values deviate from ~1.0 0.1 s-1 to ~3.3 0.3 s-1 for both the benzaldehyde and acetophenone derivatives (table 4.1). 186

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid Table 4.1. Kinetic parameters for PAM-depende nt oxidation of su bstrates. Each compound was assayed by observing the rate of oxygen consumption from a constant oxygen tension using identical reaction conditions for each ( see methods ). Comparison of Michaelis (KM,APPARENT) and inhibition (KI,APPARENT) constants for compounds fit to both Michaelis-Menton and competitive inhibition equations (equations 4.1 and 4.3) displayed a (KI/KM)APPARENT ratio for all valu es of 0.95 0.36, 187

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid and 1.08 0.23 excluding para-methoxy benzaldehyde imino-oxy acetic acid( 4), which displayed an (KI/KM)APPARENT = (7.6 M)/(24.3 M) = 0.31 0.04 (table 4.2). Inhibition constants determined for the hydroxamic acids, O -acetylbenzohydroxamic acid ( 19) and phenylacetylamino-acetic acid ( 20 ), show an intriguing trend that has the KI increase ~10-fold as a function of the me thylene linker present in compound 20, 1.17 0.092 mM ( 19) and 12.7 1 mM ( 20)(equation 4.3 and table 4.3). The glycine-extended derivatives of compounds 19 and 20 show an inverse relationship with KM with 1.3 3.6E-02 mM for N -benzoylglycine and 0.16 4.7E0-3 mM for phenylacetylamino-acetic acid ( 72). Kinetic isotope effect analysis of two s ubstrates, acetophenone imino-oxy acetic acid ( 8) and -naphthaldehyde imino-oxy acetic acid ( 17 ) was performed under ambient oxygen saturation (table 4.3). DVMAX,APPARENT values were similar between compounds with the values 2.20 0.11 (8) and 2.84 0.19 ( 17) observed. Conversely, the D(VMAX/KM)APPARENT for C-H bond cleavage values we re ~two-fold different at 2.97 0.49 ( 8) and 6.30 1.09 (17) (table 4.3). 188

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid Table 4.2. Inhibition constant (KI) determination for imino-oxy acetic acid and hydroxamic acid compounds. For oxidizable substrates, the ratio of inhibition constant : Michaelis constant (KI/KM). Inhibition constants were determined from Dixon analysis for competitive inhibition ( see methods ). Table 4.3. Apparent -dideutero kinetic isotope effect DKIE,APP for benzaldehyde iminooxy acetic acid (1), acetophenone imino-oxy acetic acid (8) and -naphthaldehyde iminooxy acetic acid (17) measured under ambient oxygen tension (217 M). 189

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid Structure-Function Analysis This study introduces O -acetylbenzohydroxamic acid ( 19) and phenylacetylamino-oxy acetic acid ( 20) as PAM inhibitors. Structures 19 and 20 were adapted from N -glycine extended compounds to N -carboxymethylhydroxylamine analogues. Previous data has shown the Nglycine extended N -benzoylglycine and phenylacetylamino-acetic acid compounds to be effective substrates for PAM oxidation (VMAX,APPARENT = 8.2 7.9E-02 and 7.4 8.6E-02 s-1, KM,APPARENT = 1.3 3.6E-02 and 0.16 4.7E-03mM)( 72). The KI,APPARENT for these compounds is 1.17 0.09 mM ( 19) and 12.7 1.0 mM ( 20) (table 4.2) and were not observed to consume oxygen under standard conditions, thus excluding KM,APPARENT calculation. A direct comparison of free glycine to carboxymethylhydroxylamine ( 16) showed that only the latter was a substrate for PAM, displaying a VMAX,APPARENT of 1.19 0.03 s-1 and a KM,APPARENT of 5.90 0.60 mM, with a (VMAX/KM),APP of 2.01E+02 2.11E+01 s-1M-1 respectively. Electronic Structure Calculations: Compounds docked within the reduced PH M active site were analyzed by both MEP and NPA to provide information a bout chemical reactivity using these nonequilibrium geometries (figure 4 .5). Please note, the ME P renderings in figure 4.5 show all molecules normalized to the same electrostatic potential scale (-205 to +50 meV). Comparison of the single point energy analys is of docked geometries obtained will be considered in pairs: Glycine versus Carboxymethylhydroxylamine For carboxymethylhydroxylamine, the NPA values for nitrogen show a considerable decrease in electron repulsion (-0.888 to 0.579) compared to glycine. This is also 190

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid observed as the -carbon of carboxymethylhydroxylamin e displays decreased electron repulsion as a function of oxygen insertion decreasing from -0.350 to -0.204. For the MEP analysis of glycine the electronegativity of the carboxylate terminus was shown to be approximately equal nitrogen lone pair. For carboxymethylhydroxylamine ( 16), the interpolated electrostatic ratios from MEP showed the carboxylate to be the most electronegative, followed by a less negative nitrogen lone pair then the amino-oxy oxygen. Second-order perturbation theory values (table 4.4) show the nitrogen lone pair of glycine to be sp3.15 with no observed N-H interaction. The nitrogen lone pair of carboxymethylhydroxylamine is observed to be sp2.25 hybridized. Both oxygen lone pairs are sp1.02 which interact with each N-H in the molecule (table 4.4, blue ). N-Benzoylglycine versus O-a cetylbenzohydroxamic Acid The oxygen lone pair in O -acetylbenzohydroxamate (19) appears to decrease the nitrogen lone pair delocalization into the carbonyl anti -bond with stabilization energies decreasing from 59.87 kcal/mol to 11.95 and 22.95 kcal/mol, respectively. This appears to occur as the oxygen lone pair stabilizes the N-H. The result is a decrease in nitrogen hybridization of the N-H from sp3.06 to sp2.18 (table 4.4, blue ). For O acetylbenzohydroxamic acid, NPA shows that oxygen insertion decreases the electron repulsion observed at the nitr ogen from -0.607 to -0.301 (Figure 4 .5). This oxygen also slightly increases the electr onegativity at the adjacent NH hydrogen from 0.423 to 0.415. Consistent with carboxymethylhydroxylamine ( 16), the inserted oxygen also decreases electron repulsion in the C of O -acetylbenzohydroxamic acid ( 19). The carbonyl moiety is also altered as a function of oxygen insertion, decreasing at the oxygen from 191

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid 0.674 to -0.631 and electron re pulsion at the carbon increa ses as a function of oxygen insertion from 0.613 to 0.570. Electron repulsion at the -carbon hydrogens were decreased from 0.234/0.266 to 0.221/0.199 with oxyge n insertion. All other aspects of each molecule were constant, irrespective of oxygen insertion. MEP analysis showed the carboxylate of N -benzoylglycine to be the most elec tronegative species in the molecule followed by the amide oxygen then nitrogen, respectively. The O -acetylbenzohydroxamic acid ( 19) also displayed the greatest electronega tivity in the carboxyl ate moiety, though differences were observed as the next most electronegative species which followed the carbonyl was the O -acetyl hydroxamic acid oxygen, followed by the nitrogen, respectively. Phenylacetylamino-acetic Acid versus Phenylacetylamino-oxy acetic Acid Each oxygen lone pair of phenylacetylamino-oxy acetic acid ( 20) stabilizes the N-H (4.31 and 3.96 kcal/mol). The nitrogen of this bond displays a decrease in hybridization from sp3.06 in the phenylacetylamino-acetic acid sp2.08 in phenylacetylamino-oxy acetic acid(20 ). The methylene linker alters the amide to hydroxamic acid electron repulsion trends sim ilar to those observed with the previous pairing. Oxygen insertion decreases the electron repulsion of the nitrogen (-0.630 to -0.299), though there is a negligible eff ect on the N-H hydrogen (0.412 versus 0.414). The carbonyl moiety altered by oxygen insertion, though the carbonyl oxygen of each derivative display very close NPA va lues (-0.679 versus -0.681), though the carbon in the hydroxamic acid derivative ( 20) displays more electron repulsion from 0.630 to 0.602, respectively. The alpha-carbon shows a decrease in elec tron repulsion as a function of oxygen insertion (-0.370 versus -0.221). Interestingly, only one of the C 192

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid hydrogens were significantly changed w ith oxygen insertion, increasing electron repulsion in the hydroxamic acid derivative ( 20) from 0.206 to 0.275. MEP analysis for phenylacetylamino-acetic acid, as expected, display the carboxylate terminus as the most electronegative moiety on the molecule closely followed by the amide oxygen then nitrogen, respectively. In good agreemen t with NPA, oxygen insertion within phenylacetylamino-oxy acetic acid (20) shows the carboxylate terminus to contain the greatest electronegativ ity followed by the carbonyl oxygen of the hydroxamoyl moiety, the O -acetyl-linked oxygen displayed a slightly greater electronegativity than the adjacent nitrogen. (Benzylidene-amino)-Acetic Acid versus Benzaldehyde Imino-Oxy Acetic Acid The nitrogen lone pairs for each molecule sh ow a high degree of stabilization into the aldimine C-H *, 10.31 and 7.27 kcal/mol respectively. The decrease in nitrogen hybridization from sp1.75 to sp1.18 through oxygen insertion suggests that the oxygen lone pair delocalize strongly into the of the imine (27.61 kcal/mol). NPA of this pair was done to directly compare the effect the oximate oxygen has on the occupancy of the imine. The aldimine hydrogen of the oxime derivative display decreased electron repulsion (0.205 versus 0.175). The adjacent carbon displays an essentially neutral occupancy for each molecule (0.005 and -0.054). As expected, the oxime oxygen decreases the electronic occupancy in the C from -0.389 to -0.210 and a relatively small effect on the C hydrogens 0.207/0.239 and 0.218/0.228, respectively. The nitrogen is drastically altered in the oxime derivative decreasing from -0.363 to -0.0956. The MEP of (benzylidene-amino)-acetic acid shows th e carboxylate moiety to be the most electronegative, while the imine displays a strong gradient from moderately 193

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid electronegative at the nitrogen to an neutra l green value. Benzaldehyde imino-oxy acetic acid( 1 ) also has the most electronegative group displayed as the carboxylate moiety while the oximyl oxygen followed as moderately el ectronegative, and the nitrogen and imine carbon display an electronega tive trend moving towards neut rality (light green). -205 meV +50 meV -205 meV +50 meV Glycine -0.888 0.343 0.366 0.183 0.207 -0.350 0.692 -0.770 -0.782 -0.888 0.343 0.366 0.183 0.207 -0.350 0.692 -0.770 -0.782 -205 meV +50 meV -205 meV +50 meV Carboxymethylhydroxylamine -0.781 -0.774 0.676 0.201 0.199 -0.430 0.366 0.326 -0.579 -0.204 -0.781 -0.774 0.676 0.201 0.199 -0.430 0.366 0.326 -0.579 -0.204 -205 meV +50 meV -205 meV +50 meV N -Benzoylglycine (Hippurate) -0.755 -.780 0.700 -0.674 -0.607 0.423 0.613 -0.373 0.234 0.266 -0.122 0.231 -0.155 0.228 -0.248 0.224 -0.234 0.214 -0.223 0.224 -0.180 -0.755 -.780 0.700 -0.674 -0.607 0.423 0.613 -0.373 0.234 0.266 -0.122 0.231 -0.155 0.228 -0.248 0.224 -0.234 0.214 -0.223 0.224 -0.180 194

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid -205 meV +50 meV -205 meV +50 meV O -Acetylbenzohydroxamic Acid -0.752 -0.769 0.669 0.221 0.199 -0.210 -0.351 -0.301 0.415 -0.631 0.570 -0.124 0.226 -0.170 0.231 -0.242 0.227 -0.242 -0.219 0.215 0.223 -0.184 -0.752 -0.769 0.669 0.221 0.199 -0.210 -0.351 -0.301 0.415 -0.631 0.570 -0.124 0.226 -0.170 0.231 -0.242 0.227 -0.242 -0.219 0.215 0.223 -0.184 -205 meV +50 meV -205 meV +50 meV Phenylacetylamino-acetic acid -0.766 -0.768 0.680 0.222 0.275 -0.370 -0.601 0.414 -0.679 0.630 0.269 0.236 -0.554 0.011 0.211 -0.213 0.210 -0.219 0.224 -0.252 0.233 -0.229 0.233 -0.200 -0.766 -0.768 0.680 0.222 0.275 -0.370 -0.601 0.414 -0.679 0.630 0.269 0.236 -0.554 0.011 0.211 -0.213 0.210 -0.219 0.224 -0.252 0.233 -0.229 0.233 -0.200 -205 meV +50 meV -205 meV +50 meV Phenylacetylamino-oxy acetic acid 0.227 0.206 -0.221 0.678 -0.772 -0.761 -0.347 0.412 -0.299 -0.681 0.602 0.286 -0.559 0.235 -0.004 0.214 -0.223 0.213 -0.218 0.224 -0.249 0.227 -0.240 -0.223 0.214 0.227 0.206 -0.221 0.678 -0.772 -0.761 -0.347 0.412 -0.299 -0.681 0.602 0.286 -0.559 0.235 -0.004 0.214 -0.223 0.213 -0.218 0.224 -0.249 0.227 -0.240 -0.223 0.214 -205 meV +50 meV -205 meV +50 meV (Benzylidene-amino)-acetic acid -0.363 -0.765 -0.739 0.709 -0.389 0.239 0.207 0.005 0.175 -0.106 0.209 -0.200 0.208 -0.229 0.219 -0.254 0.228 -0.243 0.246 -0.156 -0.363 -0.765 -0.739 0.709 -0.389 0.239 0.207 0.005 0.175 -0.106 0.209 -0.200 0.208 -0.229 0.219 -0.254 0.228 -0.243 0.246 -0.156 195

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid -205 meV +50 meV -205 meV +50 meV Benzaldehyde Imino-oxy Acetic Acid -0.352 -0.748 -0.759 0.670 0.228 0.215 -0.210 -0.0956 0.205 -0.054 -0.100 0.239 -0.178 0.229 -0.237 0.221 -0.261 -0.225 0.209 -0.206 0.210 -0.352 -0.748 -0.759 0.670 0.228 0.215 -0.210 -0.0956 0.205 -0.054 -0.100 0.239 -0.178 0.229 -0.237 0.221 -0.261 -0.225 0.209 -0.206 0.210 Figure 4.5. Molecular electrostatic potential (MEP) ( left ) and natural population analysis (NPA) (right ) calculated by single point energy calculations performed on compounds docked into the peptidylglycine -hydroxylating monooxygenase (P HM) crystal structure using Quantum Polarized Ligand Docking ( QPLD ) feature of Jaguar( 69 ). Calculations were performed using the 6-31G** basis set at the B3LYP leve l of theory. Please note, red = oxygen, grey = carbon, blue = nitrogen, and white = hydrogen. 196

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid Table 4.4. Second-order perturbation theory va lues for structure-function molecules determined from natural bond orbital analysis (NBO). The interaction of lone pair electrons with neighboring antibonding orbitals is displaye d with the hybridization of lone pair donor and anti-bond acceptor as a function of interaction energy ( Eij). 197

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid Discussion Imino-Oxy Acetic Acids All tested imino-oxy acetic aci ds are oxidized by PAM. The (VMAX/KM),APP for these compounds are all greater than N -benzoylglycine (~103 s-1M-1) and similar to phenylacetylamino-acetic acid (~104 s-1M-1). Therefore, the catalytic efficiency of these compounds were very good, with KM,APPARENT values for each lower than either aforementioned glycine-extended substrate. Strong correlati on between Michaelis constant and inhibition constant values suggest that the imino-oxy acetic acids are competitive inhibitors of glycine-extended subs trates and that binding occurs only in the active site of PAM (table 4.2). Comparison of the D(VMAX/KM),APPARENT for acetophenone-( 8) and -naphthaldehyde imino-oxy acetic acid( 17 ) to benzaldehyde imino-oxy acetic acid (1 ) showed the latter to display an increase magnitude from 6.30 1.09 ( 17) versus 4.86 0.58 (1) (table 4.3). This showed that under ambient oxygen conditions, the rate limitation of C-H cl eavage increased with the more hydrophobic naphthaldehyde imino-oxy acetic acid ( 17) substrate. Altering the substrate from a benzaldehyde (1) to acetophenone ( 8 ) derivative lowered the D(VMAX/KM),APPARENT for CH bond cleavage from 4.86 0.58 ( 1 ) to 2.97 0.49 ( 8). These results suggest that the rate limitation attributed to C-H bond cleavag e is dependent on subs trate structure. Effect of Oxygen-Insertion Benzaldehyde imino-oxy acetic acid ( 1) and the carboxymethylhydroxylamine ( 16) nitrogen lone pair hybridization are more similar (sp2.25 and sp1.18) to each other than the hydroxamic acid derivatives ( 19 and 20 ), which show p-orbital occupation 198

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid predominantly (sp35.75 and p) (table 4.4). The phenylacetylamino-acetic acid and Nbenzoylglycine molecules also show pr edominant p-orbital character, with sp11.62 and sp15.35, though the corresponding hybridization character between derivatives is increased, sp35.78 and p respectively. Another inte resting aspect to oxygen insertion between carboxymethylhydroxylamine ( 16) and benzaldehyde imino-oxy acetic acid ( 1) were the delocalization patterns that arose from the oxygen lone pair. In carboxymethylhydroxylamine ( 16), both oxygen lone pairs have equivalent hybridization, sp1.02 for each. This sp~1 hybridization pattern was also observed on the benzaldehyde imino-oxy acetic acid ( 1), though for each of the hydr oxamic acid derivatives ( 19 and 20), only one oxygen lone pair met the sp~1 specification, while the other lone pair had completely p character. The hydroxamic aci d derivatives have a delocalization pattern that restricts the geometry of the hydroxama te (O-NH-C=O) moiety. This results in a decreased amide (NH-C=O) delocaliza tion that was characteristic of the phenylacetylamino-acetic acid and benzoyl-glycine substrates. The oxime moiety is electronically analogous to the amide moiety as the nitrogen lone pair is observed to delocalize directly in to the aldimini c hydrogen and the oxygen lone pair delocalizes directly into the imine (C=N) bond. This is very similar to Nbenzoylglycine as the lone pair electrons on the amide oxygen in teract with anti-bonds of the N C of the amide, and the nitrogen lone pair interacts with the N H and strongly delocalizes into the carbonyl (C=O) moiety. Similarly, hyperconjuga tive stabilization for the of the imine (0.6853*(p)N8 0.7282*(p)C9 = 27.61 kcal/mol, table 4.4) and of the adjacent C-H aldimine bond (0.6250*(sp2.29)C9 0.7806*(s)H21 = 7.27 kcal/mole, table 4.4) of benzaldehyde imino-oxy acetic acid ( 1 ) by oxygen lone pairs and also 199

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid provide electronic rationale for the preference of thes e compounds to be in the Eisomer( 56). What differs between the oxime and amide is a singly bonded oxygen which delocalizes into an imine and the imine nitrogen lone pair delocalizes into the aldiminic hydrogen for the benzaldehyde imino-oxy acetic acid, while the oxygen lone pairs of the amide delocalize into the C N bond, the nitrogen lone pair delocalizes into both the carbonyl and N H *-anti-bonds. Both species share stro ng similarities electronically, as both carbonyl and imine moieties are stabili zed by lone pair inte raction from their adjacent atom. Interestingly, the nitrogen lone pair of both species stabilizes a hydrogen atom, for the amide the N-H and for the aldiminic hydrogen for the aldoxime (figure 4 .6). O C N H N C O H =Amide Oxime Figure 4.6. Comparison of lone pair -antibond interaction for am ide versus oxime moiety derived from natural bond orbital analysis. As listed in table 4.4, the delocalization of carbonyl oxygen lone pairs into adjacent N-H bond of the amide and hydroxamate moieties are super-imposable displaying lone pair hybridizations of sp~0.4 and full p orbital, respectively, with corresponding delocalization energies into th e adjacent N-C bonds are also within error, ~1.18-to-1.85 and 14.13-to-17.05 kcal/mol, resp ectively. This suggests that the 200

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid inserted oxygen only affect the electronic prope rties of the nitrogen. This was alluded to with a mild decrease in the hybridization ch aracter of the bonds for the glycine-extended and hydroxamic acid derivatives. The anti-bond interaction of the N-C bond was 0.6*(sp~1.8)N-0.78*(sp~2.1)C for the amide and 0.6*(sp~1.5)N-0.78*(sp~2.25)C for the hydroxamic acid derivatives. From this, it was observed that although the hybrid composition (value in front of each parent hesis) was constant between moieties, the actual sp hybridization subtly decreased fo r nitrogen and increased for carbon as a function of oxygen insertion. Although, this may appear counter intuitive, it actually begins to lay the framework for delocaliza tion of the inserted oxygen into the N-H and the adjacent effect on the carbonyl in the hydroxamic acid derivatives. Also, carbonyl oxygen lone pair delocalizati on into the adjacent N-C bond was comparable with each of the hydroxamates ( 19 and 20) to their glycine-extended de rivatives. Specifically, 15.17 & 1.82 kcal/mole N -benzoylglycine versus 14.13 & 1.79 kcal/mole for Oacetylhydroxamic acid and phenylacetylamino-a cetic acid 1.82 & 15.17 kcal/mole versus phenylamino-oxy acetic acid 1.18 & 17.05 kcal/mole, respectivel y (table 4.4). Thus, when an inserted oxygen was present, the N H *-anti-bond orbital interaction became stabilized, increasing the sp -hybridization at the adjacent carbonyl carbon (figure 4 .7). R O N H O O OH R O N H O OH R =0 = N -Benzoylglycine R =1 = Phenylacetylamino-acetic acid R =0 = O -Acetylhydroxamic Acid (19) R =1 = Phenylacetylamino-oxyacetic acid (20) Figure 4.7. Alteration in delocalization pattern as a function of oxygen insertion into N benzoylglycine and phenylacetylamino-acetic acid as predicted by NBO analysis (table 4.4). 201

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid This trend differs with consider ation of carboxymethylhydroxylamine ( 16) as a substrate because the N H hybridization is sp~3 while in the hydroxamic acid derivatives (19 and 20 ) it is sp~2 (table 4.4, shown in blue ). Also, the lone pair hybridization of the nitrogen is much increased compared to the glycine-extended molecules. Together, oxygen in sertion perturbs the standa rd amide-type delocalization pattern, by interacting di rectly with the N-H bond creating a pseudo-double bond moiety. The data suggest that there is a requ irement for lone pair delocalization adjacent to the -carbon for these molecules to be substrates. The effect of oxygen insertion into the free glycine molecule (carboxymethylhydroxylamine ( 16)) was an unexpected PAM substrate, whereas glycine was not (up to [glycine] = 1M). The oxygen lone pair perturbs the carboxymethylhydroxylamine ( 16) differently than the hydroxamic acid compounds. The required interaction appears to be the sp~3 for the nitrogen (N-H) bond. The stabilization effect is observed through oxyge n lone pair interact ion into these bonds, respectively (figure 4 .8). N H H O O OH Figure 4.8. Delocalization pattern for carboxymethylhydroxylamine ( 16) as predicted by NBO analysis (table 4.4). Conclusion It appears that the benzal dehyde imino-oxy acetic acid is electronically similar to N -benzoylglycine. Oxygen insertion into N -benzoylglycine appears to disrupt the amide delocalization through a anti-bond interaction with the N H bond. The reason this affects oxidation is difficult to reconcile clearly, though it may appear that a strong 202

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid delocalization event adjacent to the C bond is required for substrate activation. The insertion of oxygen disrupts this requirement wi th a weak delocalization into the adjacent N H -anti-bonding orbital, though more impor tantly it appears to disrupt the sphybridization of this N H bond. Though, if it were only an issue of weak delocalization into an adjacent bond as s een in the hydroxamic acid compounds (figure 4.7), then carboxymethylhydroxylamine (16) would not be oxidized. The fact that it is a substrate, suggests that th e sp-hybridization of the N H bond is a critical factor determining whether oxygen insertion yields a non-productive substrate. As a counterpoint, the sp-hybridizat ion of the inserted oxygen may also play a critical role (table 4.4). This may be a semantic argumen t, as the increase in p-character directly accompanies the decrease in the sp-hybridization of the adjacent nitrogen. For oxygencontaining substrates, carboxymethylhydroxylamine ( 16) and benzaldehyde imino-oxy acetic acid ( 1), the sp-hybridization for each of the oxygen lone pair orbitals is sp~1 which suggests that both oxygen lone pairs and the N H anti-bonding environment play an equally critical role in determining the difference between substrate activation and competitive inhibition. Overall, this sugge sts that static docking poses are not enough to distinguish between substrates versus non-substrates for PHM. Both the docked substrate orientation and electrost atic potential calculations do not correlate to any discernable structure-function trend observed. Conversely, subtle contributions from hyperconjugative stabilization do provide an informative theoretical framework to improve current understanding of substrate react ivity in PHM. This study demonstrated that consistent trends th at arise from lone pair* interactions allow predictions concerning reactivity to be directly addresse d. The application of NBO analysis 203

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid allowed trends in lone pairan ti-bonding orbital inte raction to be used to rationalize the unexpected activity of the imino-oxy acetic acid and carboxymethylhydroxylamine ( 16) and inactivity of free-glyc ine and the hydroxamic acid ( 19 and 20) as PAM substrates. The framework from which these results were derived is the first application of NBO analysis to an enzymatic structure-function st udy. Application of NBO to the suicide inhibitor 4-phenyl-3-butenoic acid (PBA) would determine the role hyperconjugative stabilization of the C-H has on the partition ratio. By understanding the relationship the C-H has on substrate and inactivator behavi or would allow the partition ratio of PBA could become a malleable trait. The oxidation of all synthesized iminooxy acetic acids by PAM was interesting as many of these derivatives had previously showed anti-in flammatory activity coupled to low toxicity in rats( 56 ). As discussed in the intr oduction, chronic inflammation is associated with the PAM-dependant activati on of Substance P and CGRP, respectively. As all tested imino-oxy acetic acids showed a lower KM,APP values than either glycineextended compound observed, the oxime moiety may be preferred to the amide for effective binding into the active-site of PHM. This structural preference for the oxime by PAM was further shown as (VMAX/KM),APP values were oxidized as or more efficiently than either N -benzoylglycine or phenylacetylamino-acetic acid. Therefore, the mechanistic data produced with benzaldehyde imino-oxy acetic acid ( 1) lend a considerable amount of understa nding to the kinetic behavior during oxidation while the structure-function study provided electronic structure information. Future work with these compounds should utilize the methodologi es presented to construct and understand 204

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid potent mechanism-based inhibitors based on oxime containing compounds to efficiently target PAM. Potential Applications of NBO Analysis Structure-function data for PAM must be thoroughly evaluated in the design of novel neuropeptide analogues that have potenti al pharmacological interest. Using NBO analysis to supplement kinetic analysis and highly accurate docking algorithms provided a novel route to rationalize the contributio n subtle electronic differences have on structure-function relationships. For example, in the absence of NBO analysis, the electronic similarity between N -benzoylglycine and benzal dehyde imino-oxy acetic acid ( 1) could not have been determ ined from kinetic, docking or MEP analysis. Therefore, NBO analysis delivers essential electronic st ructure information that allows a coherent picture of previously separa te components used in structure-function studies. This information would be a useful aid to any medicinal chemist searching for ultimate specificity for their target. From a more philosophical pers pective, NBO analysis will likely be a fundamental concept to unde rstand Lewis and non-Lewis bonding within proteins to uncover the chemical rationale f acilitating their folding. Advances using this technique may allow synthetic proteins to fully mimic native tertiary structures. Perhaps one of the next great advances in synthetic chemistry will be the growth from functional group protection to peptide fold ing groups which guide macrom olecules toward a desired and stable orientation. This will drastically alter how enzy mologists research chemical mechanism and will open an entire field to pr otein therapy. An efficient automation of this process would be an incred ible advance to all fields us ing proteins; its impact would certainly merit the highest honor in chemistry, the Nobel Prize. 205

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid Experimental benzaldehyde imino-oxy acetic acid (1) : 1H NMR analysis (250MHz, MeODd4) 4.96 (singlet, 2H, CH2, -methylene), 7.66 (m, 3H, ArH), 7.87 (m, 2H, ArH), 8.48 (singlet, 1H, H-C=N). 13C NMR analysis (62.5MHz, MeODd4) 171.841 (C=O,carboxylic acid), 149.486 (C=N, imine), 130.911 (Ar, C-1), 129.188 (Ar, C-4), 127.673 (Ar, C-3, C-5), 126.136 (Ar, C-2, C-6), 69.325 (CH2, -methylene), m.p. (93-94C). para-cyano benzaldehyde imino-oxy acetic acid (2) : 1H NMR analysis (250MHz, MeODd4) 4.96 (singlet, 2H, CH2, -methylene), 7.66 (m, 3H, ArH), 7.87 (m, 2H, ArH), 8.48 (singlet, 1H, H-C=N). 13C NMR analysis (62.5MHz, MeODd4) 171.841 (C=O,carboxylic acid), 149.486 (C=N, imine), 130.911 (Ar, C-1), 129.188 (Ar, C-4), 127.673 (Ar, C-3, C-5), 126.136 (Ar, C-2, C-6), 69.325 (CH2, -methylene), m.p. (93-94C). para-nitro benzaldehyde imino-oxy acetic acid (3) : 1H NMR analysis (250MHz, MeODd4) 4.36 (singlet, 2H, CH2, -methylene), 7.35 (m, 2H, ArH), 7.73 (m, 2H, ArH), 7.84 (singlet, 1H, H-C=N). 13C NMR analysis (62.5MHz, MeODd4) 171.297 (C=O,carboxylic acid), 147.640 (C=N, imine), 147.407 (Ar, C-1), 137.112 (Ar, C-4), 126.902 (Ar, C-3, C-5), 126.772 (Ar, C-2, C-6), 69.728 (CH2, -methylene), m.p. (140-2C). para-methoxy benzaldehyde imino-oxy acetic acid (4) : 1H NMR analysis (250MHz, MeODd4) 3.38 (singlet, 2H, CH3O), 4.29 (singlet, 2H, CH2, -methylene), 6.50 (m, 206

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid 2H, ArH), 7.11 (m, 2H, ArH), 7.71 (singlet, 1H, H-C=N). 13C NMR analysis (62.5MHz, MeODd4) 171.872 (C=O,carboxylic acid), 160.594 (C=N, imine), 149.039 (Ar, C-1), 127.674 (Ar, C-4), 123.411 (Ar, C-3, C-5), 113.066 (Ar, C-2, C6), 69.143 (CH2, -methylene). para-chloro benzaldehyde imino-oxy acetic acid (5) : 1H NMR analysis (400MHz, DMSOd6) 4.648 (singlet, 2H, CH2, -methylene), 7.450 (m, 2H, ArH), 7.619 (m, 2H, ArH), 8.331 (singlet, 1H, H-C=N). 13C NMR analysis (100MHz, DMSOd6) 171.565 (C=O,carboxylic acid), 149.458 (C=N, imine), 135.413 (Ar, C-1), 131.156 (Ar, C-4), 129.620 (Ar, C-2, C-3, C-5, C-6), 71.153 (CH2, -methylene), m.p. (1235C). para-fluoro benzaldehyde imino-oxy acetic acid (6) : 1H NMR analysis (400MHz, DMSOd6) 4.617 (singlet, 2H, CH2, -methylene), 7.259 (m, 2H, ArH), 7.663 (m, 2H, ArH), 8.319 (singlet, 1H, H-C=N). 13C NMR analysis (100MHz, DMSOd6) 171.607 (C=O,carboxylic acid), 149.439 (C=N, imine), 129.968 (Ar, C-3, C-5), 116.717 (Ar, C-2, C-6), 71.061 (CH2, -methylene). para-hydroxyl benzaldehyde imino-oxy acetic acid (7) : 1H NMR analysis (400MHz, DMSOd6) 4.572 (singlet, 2H, CH2, -methylene and 4-OH), 6.794 (m, 2H, ArH), 7.428 (m, 2H, ArH), 8.166 (singlet, 1H, H-C=N). 13C NMR analysis (100MHz, DMSOd6) 171.843 (C=O,carboxylic acid), 160.029 (C=N, imine), 150.279 (Ar, C-1), 129.418 (Ar, C-2, C-6), 123.068 (Ar, C-4), 116.366 (Ar, C-3, C-5), 70.840 (CH2, methylene). 207

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid acetophenone imino-oxy acetic acid (8) : 1H NMR analysis (250MHz, MeODd4) 2.55 (singlet, 3H, CH3), 4.97 (singlet, 2H, CH2, -methylene), 7.63 (m, 3H, ArH), 7.91 (m, 2H, ArH). 13C NMR analysis (250MHz, MeODd4) 172.020 (C=O, carboxylic acid), 162.319 (C=N, imine), 135.202 (Ar, C-1), 128.330 (Ar, C-2, C-6), 127.295 (Ar, C4), 125.135 (Ar, C-3, C-5), 69.264 (CH2, -methylene), 11.049 (CH3), m.p. (9798C). -dideutero acetophenone imino-oxy acetic acid (8b): 13C NMR analysis (62.5MHz, MeODd4) 176.759 (C=O, carboxylic acid), 157.794 (C=N, imine), 134.752 (Ar, C-1), 128.885 (Ar, C-2, C-6), 127.717 (Ar, C-4), 125.549 (Ar, C-3, C5), 12.681 (CH3), m.p. (99-101C). para-cyano acetophenone imino-oxy acetic acid (9) : 1H NMR analysis (250MHz, MeODd4) 1.87 (singlet, 3H, CH3), 4.34 (singlet, 2H, CH2, -methylene), 7.30 (m, 2H, ArH), 7.40 (m, 2H, ArH). 13C NMR analysis (62.5MHz, MeODd4) 172.285 (C=O, carboxylic acid), 154.675 (C=N, imine), 140.286 (Ar, C-1), 131.927 (Ar, C-2, C-6), 126.575 (Ar, C-4), 118.116 (Ar, C-3, C-5), 112.212 (C N, cyano), 69.264 (CH2, -methylene), 11.268 (CH3), m.p. (139-142C). para-nitro acetophenone imino-oxy acetic acid (10) : 1H NMR analysis (250MHz, D2Od2) 2.52 (singlet, 3H, CH3), 4.77 (singlet, 2H, CH2, -methylene), 7.10 (m, 2H, ArH), 7.73 (m, 2H, ArH). 13C NMR analysis (62.5MHz, MeODd4) 177.128 (C=O, carboxylic acid), 158.055 (C=N, imine), 147.457 (Ar, C-1), 127.187 (Ar, C-2, C-6), 115.022 (Ar, C-3, C-5), 71.133 (CH2, -methylene), 12.425 (CH3), m.p. (139-142C). 208

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid para-methoxy acetophenone imino-oxy acetic acid (11) : 1H NMR analysis (250MHz, MeODd4) 1.82 (singlet, 3H, CH3), 3.36 (singlet, 3H, CH3O), 4.26 (singlet, 2H, CH2, -methylene), 6.46 (m, 2H, ArH), 7.14 (m, 2H, ArH). 13C NMR analysis (62.5MHz, MeODd4) 171.935 (C=O, carboxylic acid), 160.213 (C=N, imine), 155.155 (Ar, C4), 127.668 (Ar, C-1), 126.502 (Ar, C-2, C-6), 112.405 (Ar, C-3, C-5), 69.126 (CH2, -methylene), 53.602 (CH3O), 10.882 (CH3), m.p. (105-106C). para-chloro acetophenone imino-oxy acetic acid (12) : 1H NMR analysis (250MHz, MeODd4) 1.82 (singlet, 3H, CH3), 4.30 (singlet, 2H, CH2, -methylene), 6.92 (m, 2H, ArH), 7.19 (m, 2H, ArH). 13C NMR analysis (62.5MHz, MeODd4) 171.772 (C=O, carboxylic acid), 154.371 (C=N, imine), 134.169 (Ar, C-4), 133.792 (Ar, C-1), 127.402 (Ar, C-2, C-6), 126.593 (Ar, C-3, C-5), 69.331 (CH2, -methylene), 10.740 (CH3), m.p. (119-122C). para-fluoro acetophenone imino-oxy acetic acid (13) : 1H NMR analysis (250MHz, MeODd4) 1.87 (singlet, 3H, CH3), 4.36 (singlet, 2H, CH2, -methylene), 6.923 (m, 2H, ArH), 7.17 (m, 2H, ArH). m.p. (104-107C). para-hydroxy acetophenone imino-oxy acetic acid (14) : 1H NMR analysis (250MHz, MeODd4) 2.60 (singlet, 3H, CH3), 5.03 (singlet, 2H, CH2, -methylene), 7.13 (m, 2H, ArH), 7.87 (m, 2H, ArH). 13C NMR analysis (62.5MHz, MeODd4) 173.226 (C=O, carboxylic acid), 158.526 (C=N, imine), 156.643 (Ar, C-4), 127.618 (Ar, C-2, C-6), 115.010 (Ar, C-3, C-5), 70.003 (CH2, -methylene), 11.986 (CH3), m.p. (142145C). 209

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid para-amino acetophenone imino-oxy acetic acid (15) : 13C NMR analysis (100MHz, DMSOd6) 172.237 (C=O, carboxylic acid), 155.995 (C=N, imine), 150.568 (Ar, C4), 127.716 (Ar, C-2, C-6), 123.506 (Ar, C-1), 114.122 (Ar, C-3, C-5), 70.739 (CH2, -methylene), 12.938 (CH3), m.p. (152-155C). beta-naphthaldehyde imino-oxy acetic acid (17) : 1H NMR analysis (400MHz, DMSOd6) 4.66 (singlet, 2H, CH2, -methylene), 7.53 (m, 2H, C-5, C-6 ArH), 7.73 (m, 1H, C8 ArH), 7.90 (doublet, 4H, C-4, C-7, C-9, C-10 ArH), 8.05 (singlet, 1H, H-C=N), 8.42 (singlet, 1H, C2 ArH). 13C NMR analysis (100MHz, DMSOd6) 171.614 (C=O, carboxylic acid), 150.524 (C=N, imine), aromatic region: 134.325, 133.389, 129.876, 129.189, 128.448, 127.879, 127.509, 123.289, 71.164 (CH2, methylene), m.p. (132-133C). -dideutero naphthaldehyde imino-oxy acetic acid (17b) 1H NMR analysis (400MHz, DMSOd6) 7.53(m, 2H, C-5, C-6 ArH), 7.73 (m, 1H, C8 ArH), 7.91 (doublet, 4H, C4, C-7, C-9, C-10 ArH), 8.06 (singlet, 1H, H-C=N), 8.48 (singlet, 1H, C2 ArH), m.p. (131-132C). para-(5-Dimethylaminonapthalene-1-sulfo namido)acetophenone imino-oxy acetic acid (18) 1H NMR analysis (400MHz, DMSOd6) 1.81 (singlet, 3H, C(O)-CH3, methyl), 2.51 (singlet, 6H, CH3-NR2, methyl), 4.29 (singlet, 2H, CH2, -methylene), 6.79 (m, 2H, naphthalene, C-3, C-4), 6.97 (m, 1H, naphthalene, C-5), 7.16 (m, 2H, C-2, C-6, ArH), 7.33 (m, 2H, C-3, C-5, ArH), 7.94 (m, 1H, naphthalene, C-5), 8.15 (m, 2H, naphthalene, C-3, C-4), 10.56 (singlet, 1H, carboxylic acid). 13C NMR analysis (100MHz, DMSOd6) 171.002 (C=O, carboxylic acid), 155.995(C=N, imine), 210

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid 150.568 (Ar, C4 NH), 138.753 (Dansyl, C-6), 134.534 (Dansyl, C-1), 130.226 (Dansyl, C-2), 129.913 (Dansyl, C-7), 128.935 (Dansyl, C-8), 128.267 (Dansyl, C-4 and C-9), 127.716 (Ar, C-3, C-5), 123.506 (Ar, C-4), 118.564 (Dansyl, C-3 and C10), 115.360 (Dansyl, C-5), 114.122 (Ar, C-2, C-6), 70.231 (CH2, -methylene), 45.051 (N(CH3)2, di-methylamine), 12.938 (CH3, ketimine), m.p. (156-158C). 211

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid References (1) Prigge, S. T., Mains, R. E., Eipper, B. A., and Amzel, L. M. (2000) New insights into copper monooxygenases and peptide amidation: structure, mechanism and function. Cell Mol Life Sci 57, 1236-59. (2) Walsh, C. T. (2006) Posttranslational Modifications of Proteins: Expanding Nature's Inventory, Roberts and Company Publishers, Englewood, Colorado. (3) Bolkenius, F. N., and Ganzhorn, A. J. (1998) Peptidylglycine alpha-amidating mono-oxygenase: neuropeptide amidation as a target for drug design. Gen Pharmacol 31, 655-9. (4) Saldise, L., Martinez, A., Montuenga, L. M., Treston, A., Springall, D. R., Polak, J. M., and Vazquez, J. J. (1996) Distri bution of peptidyl-glycine alpha-amidating mono-oxygenase (PAM) enzymes in norma l human lung and in lung epithelial tumors. J Histochem Cytochem 44, 3-12. (5) Oldham, C. D., Li, C., Girard, P. R ., Nerem, R. M., and May, S. W. (1992) Peptide amidating enzymes are present in cultured endothelial cells. Biochem Biophys Res Commun 184 323-9. (6) Klinman, J. P. (2006) The coppe r-enzyme family of dopamine betamonooxygenase and peptidylglycine alpha-hydroxylating monooxygenase: resolving the chemical pathwa y for substrate hydroxylation. J Biol Chem 281, 3013-6. (7) Merkler, D. J. (1994) C-terminal amidated peptides: production by the in vitro enzymatic amidation of glycine-extende d peptides and the importance of the amide to bioactivity. Enzyme Microb Technol 16, 450-6. (8) Merkler, D. J., Chew, G. H., Gee, A. J., Merkler, K. A., Sorondo, J. P., and Johnson, M. E. (2004) Oleic acid derived metabolites in mouse neuroblastoma N18TG2 cells. Biochemistry 43, 12667-74. (9) Wilcox, B. J., Ritenour-Rodgers, K. J., A sser, A. S., Baumgart, L. E., Baumgart, M. A., Boger, D. L., DeBlassio, J. L., deLong, M. A., Glufke, U., Henz, M. E., King, L., 3rd, Merkler, K. A., Patterson, J. E ., Robleski, J. J., Vederas, J. C., and Merkler, D. J. (1999) N-acylglycine amid ation: implications for the biosynthesis of fatty acid primary amides. Biochemistry 38, 3235-45. (10) Rush, R. A., and Geffen, L. B. (1980) Dopamine beta-hydroxylase in health and disease. Crit Rev Clin Lab Sci 12, 241-77. (11) Groves, J. T. (2006) High-valent ir on in chemical and biological oxidations. J Inorg Biochem 100, 434-47. (12) Knaggs, S., Malkin, H., Osborn, H. M., Williams, N. A., and Yaqoob, P. (2005) New prodrugs derived from 6-aminodopami ne and 4-aminophenol as candidates for melanocyte-directed enzy me prodrug therapy (MDEPT). Org Biomol Chem 3, 4002-10. (13) Padgette, S. R., Wimalasena, K., Herman, H. H., Sirimanne, S. R., and May, S. W. (1985) Olefin oxygenation and N-dealkylation by dopamine betamonooxygenase: catalysis and mechanism-based inhibition. Biochemistry 24, 5826-39. 212

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid (14) Padgette, S. R., Herman, H. H., Han, J. H., Pollock, S. H., and May, S. W. (1984) Antihypertensive activities of phenyl ami noethyl sulfides, a class of synthetic substrates for dopamine beta-hydroxylase. J Med Chem 27, 1354-7. (15) Herman, H. H., Pollock, S. H., Padgette, S. R., Lange, J. R., Han, J. H., and May, S. W. (1983) Effects of phenyl-2-ami noethyl sulfide, a novel dopamine-betahydroxylase substrate, on the cardiovascul ar system of the anesthetized dog. J Cardiovasc Pharmacol 5, 725-30. (16) May, S. W., Phillips, R. S., Mueller, P. W., and He rman, H. H. (1981) Dopamine beta-hydroxylase. Demonstration of enzymatic ketonization of the product enantiomer S-octopamine. J Biol Chem 256, 2258-61. (17) Katopodis, A. G., Ping, D., and May, S. W. (1990) A novel enzyme from bovine neurointermediate pituitary catalyzes dealkylation of alpha-hydroxyglycine derivatives, thereby functioning seque ntially with peptidylglycine alphaamidating monooxygenase in peptide amidation. Biochemistry 29, 6115-20. (18) Katopodis, A. G., and May, S. W. (1990) Novel substrates and inhibitors of peptidylglycine alpha-amidating monooxygenase. Biochemistry 29, 4541-8. (19) Rocchi, P., Boudouresque, F., Zamora, A. J., Muracciole, X., Lechevallier, E., Martin, P. M., and Ouafik, L. (2001) Expression of adrenomedullin and peptide amidation activity in human prostate cancer and in human prostate cancer cell lines. Cancer Res 61, 1196-206. (20) Bauer, J. D., Sunman, J. A., Foster M. S., Thompson, J. R., Ogonowski, A. A., Cutler, S. J., May, S. W., and Pollock, S. H. (2007) Anti-Inflammatory Effects of 4-Phenyl-3-butenoic Acid and 5-(Acetyl amino)-4-oxo-6-phenyl -2-hexenoic Acid Methyl Ester, Potential Inhibito rs of Neuropeptide Bioactivation. J Pharmacol Exp Ther 320, 1171-7. (21) Ogonowski, A. A., May, S. W., Moore, A. B., Barrett, L. T., O'Bryant, C. L., and Pollock, S. H. (1997) Anti-inflammatory a nd analgesic activity of an inhibitor of neuropeptide amidation. J Pharmacol Exp Ther 280 846-53. (22) Mueller, S. A., Driscoll, W. J., and Mueller, G. P. (1999) Captopril inhibits peptidylglycinealpha-hydroxylati ng monooxygenase: implications for therapeutic effects. Pharmacology 58 270-80. (23) McIntyre, N. R., Lowe, E. W., Jr., Chew G. H., Owen, T. C., and Merkler, D. J. (2006) Thiorphan, tiopronin, and related anal ogs as substrates and inhibitors of peptidylglycine alpha-amidating monooxygenase (PAM). FEBS Lett 580 521-32. (24) Cutler, S. J., DeWitt Blanton, C., Jr., Ak in, D. T., Steinberg, F. B., Moore, A. B., Lott, J. A., Price, T. C., May, S. W., and Pollock, S. H. (1998) Pharmacological evaluation of 1-(carboxymethyl)-3,5-diphenyl-2-methylbenzene, a novel arylacetic acid with potential anti-inflammatory properties. Inflamm Res 47, 31624. (25) Jeng, A. Y., Fujimoto, R. A., Chou, M., Tan, J., and Erion, M. D. (1997) Suppression of substance P biosynthesis in sensory neurons of dorsal root ganglion by prodrug esters of potent peptidylglycine alpha-amidating monooxygenase inhibitors. J Biol Chem 272, 14666-71. (26) Zabriskie, T. M., Klinge, M., Szymanski, C. M., Cheng, H., and Vederas, J. C. (1994) Peptide amidation in an inverteb rate: purification, characterization, and 213

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid inhibition of peptidylglycine alpha-h ydroxylating monooxygenase from the heads of honeybees (Apis mellifera). Arch Insect Biochem Physiol 26, 27-48. (27) Silverman, R. B. (2002) The organic chemistry of enzyme-catalyzed reactions Academic Press. (28) Fitzpatrick, P. F., and Villafranca, J. J. (1987) Mechanism-based inhibitors of dopamine beta-hydroxylase. Arch Biochem Biophys 257, 231-50. (29) Fitzpatrick, P. F., and Villafranca, J. J. (1986) The mechanism of inactivation of dopamine beta-hydroxylase by hydrazines. J Biol Chem 261, 4510-8. (30) Fitzpatrick, P. F., Flory, D. R., Jr., a nd Villafranca, J. J. (1985) 3-Phenylpropenes as mechanism-based inhibitors of dopa mine beta-hydroxylase: evidence for a radical mechanism. Biochemistry 24 2108-14. (31) Rajashekhar, B., Fitzpatrick, P. F., Colombo, G., and Villafranca, J. J. (1984) Synthesis of several 2-substituted 3-(p-hydroxyphenyl)-1-p ropenes and their characterization as mechanism-based inhi bitors of dopamine beta-hydroxylase. J Biol Chem 259, 6925-30. (32) Driscoll, W. J., Konig, S., Fales, H. M., Pannell, L. K., Eipper, B. A., and Mueller, G. P. (2000) Peptidylgl ycine-alpha-hydroxylat ing monooxygenase generates two hydroxylated pr oducts from its mechanismbased suicide substrate, 4-phenyl-3-butenoic acid. Biochemistry 39, 8007-16. (33) Mueller, G. P., Driscoll, W. J., and Eipper, B. A. (1999) In vivo inhibition of peptidylglycine-alpha-hydroxylating monooxygenase by 4-phenyl-3-butenoic acid. J Pharmacol Exp Ther 290, 1331-6. (34) Northrop, D. B. (1981) The expression of isotope effects on enzyme-catalyzed reactions. Annu Rev Biochem 50, 103-31. (35) Casara, P., Ganzhorn, A., Philippo, C., Chanal, M. C., and Danzin, C. (1996) Unsaturated thioacetic acids as nove l mechanism-based inhibitors of peptidylglycine -hydroxylating monooxygenase. Bioorg. Med. Chem. Lett. 6, 393-396. (36) Bolkenius, F. N., Ganzhorn, A. J., Chan al, M. C., and Danzin, C. (1997) Selective mechanism-based inactivation of pe ptidylglycine alpha-hydroxylating monooxygenase in serum and heart atrium vs. brain. Biochem Pharmacol 53, 1695-702. (37) Berti, P. J., and Tanaka, K. S. E. (2002) Transition state analysis using multiple kinetic isotope effects: Mechanisms of enzymatic and non-enzymatic glycoside hydrolysis and transfer. Adv. Phys. Org. Chem. 37, 239-314. (38) Singh, V., Lee, J. E., Nunez, S., Ho well, P. L., and Schramm, V. L. (2005) Transition state structure of 5'-methy lthioadenosine/S-adenosylhomocysteine nucleosidase from Escherichia coli and its similarity to transi tion state analogues. Biochemistry 44, 11647-59. (39) Cramer, C. J. (2004) Computational chemistry: Theories and models second ed., John Wiley & Sons, Ltd., West Sussex. (40) Reed, A. E., L.A., C., and Weinhold, F. (1988) Intermolecular interactions from a natural bond orbital, don or-acceptor viewpoint. Chem. Rev. 88, 899-926. (41) King, B. F., and Weinhold, F. (1995) Structure and spectroscopy of (HCN)n clusters: Cooperative and electro nic delocalization effects in C-H...N hydrogen bonding. J. Chem. Phys. 103, 333-347. 214

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid (42) Weinhold, F., and Landis, C. R. (2005) Valency and Bonding: A natural bond orbital donor-acceptor perspective Cambridge University Press. (43) Pophristic, V., and Go odman, L. (2001) Hyperconjuga tion not steric repulsion leads to the staggered structure of ethane. Nature 411, 565-568. (44) Weinhold, F. (2001) A ne w twist on molecular shape. Nature 411, 539-541. (45) Mulliken, R. S. (1955) Electron ic Population Analysis on LCAO[Single Bond]MO Molecular Wave Functions. I J. Chem. Phys. 23, 1833-1840. (46) Mulliken, R. S. (1955) Electronic P opulation Analysis on LCAO-MO Molecular Wave Functions. III. Effects of H ybridization on Overlap and Gross AO Populations J. Chem. Phys. 23, 2338-2342 (47) Reed, A. E., Weinstock, R. B., and Weinhold, F. (1985) Natural population analysis. J. Chem. Phys. 83, 735-746. (48) Bent, H. A. (1961) An appraisal of valence-bond stru ctures and hybridization in compounds of the first row elements. Chem. Rev. 61, 275-311. (49) Solomons, T. W. G. (1996) Organic chemistry 6th ed., John Wiley & Sons, Inc., New York. (50) Carey, F. A., and Sundberg, R. J. (2000) Advanced organic ch emistry part A: Structure and mechanism 4th ed., Kluwer Academic/Plenum Publishers, New York. (51) Pophristic, V., and Go odman, L. (2001) Hyperconjuga tion not steric repulsion leads to the staggered structure of ethane. Nature 411, 565-568. (52) Goodman, L., and Gu, H. (1998) Flexing analysis of steric exchange repulsion accompanying ethane internal rotation. J. Chem. Phys. 110, 72-78. (53) Goodman, L., Gu, H., and Pophristic, V. (1999) Flexing analysis of ethane internal rotation energetics. J. Chem. Phys. 110, 4268-4275. (54) Goodman, L., Pophristic, V., and Weinhold, F. (1999) Or igin of methyl internal rotation barriers. Acc. Chem. Res. 32, 983-993. (55) Mo, Y., and Goa, J. (2007) Theoretical analysis of the rotational barrier of ethane. Acc. Chem. Res. 40, 113-119. (56) van Dijk, J., and Zwagemakers, J. M. (1977) Oxime ether derivatives, a new class of nonsteroidal anti-inflammatory compounds. J Med Chem 20, 1199-206. (57) Liu, K.-C., Shelton, B. R., and Howe, R. K. (1980) A Particularly Convenient Preparation of Benzohydroximinoyl Chlo rides (Nitrile Oxide Precursors). Journal of Organic Chemistry 45 3917-1919. (58) Himel, C. H., Aboul-Saad, W. G., an d Solang, U. K. (1971) Fluorescent analogs of insecticides and synergists. Synthesis and reactions of active-site-directed fluorescent probes. J. Agric. Food. Chem. 19, 1175-1180. (59) Francisco, W. A., Merkler, D. J., Blackburn, N. J., and Klinman, J. P. (1998) Kinetic mechanism and intrinsic isotope effects for the peptidylglycine alphaamidating enzyme reaction. Biochemistry 37, 8244-52. (60) Jones, B. N., Tamburini, P. P., C onsalvo, A. P., Young, S. D., Lovato, S. J., Gilligan, J. P., Jeng, A. Y., and Wennogle, L. P. (1988) A fluorometric assay for peptidyl alpha-amidation activity using high-performance liquid chromatography. Anal Biochem 168, 272-9. 215

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CHAPTER 4: Structure-Function of the Imino-Oxy Acetic Acid (61) Prigge, S. T., Eipper, B. A., Main s, R. E., and Amzel, L. M. (2004) Dioxygen binds end-on to mononuclear copper in a precatalytic enzyme complex. Science 304, 864-7. (62) GLIDE (2000) Schrdinger, LLC, Portland, OR. (63) QSITE (2000) Schrdinger, LLC, Portland, OR. (64) Friesner, R. A., Banks, J. L., Murphy, R. B., Halgren, T. A., K licic, J. J., Mainz, D. T., Repasky, M. P., Knoll, E. H., Sh elley, M., Perry, J. K., Shaw, D. E., Francis, P., and Shenkin, P. S. (2004) G lide: a new approach for rapid, accurate docking and scoring. 1. Method and a ssessment of docking accuracy. J Med Chem 47, 1739-49. (65) Cho, A. E., Guallar, V., Berne, B. J., and Friesner, R. (2005) Importance of accurate charges in molecular docking: quantum mechanical/molecular mechanical (QM/MM) approach. J Comput Chem 26, 915-31. (66) Becke, A. D. (1993) Density-functiona l thermochemistry. III. The role of exact exchange. J Chem Phys 98, 5648-5652 (67) Lee, C., Yang, W., and Parr, R. G. (1988) Development of the Colle-Salvetti correlation-energy formula into a f unctional of the electron density. Phys Rev B Condensed Matter 37, 785-789. (68) Lawrence, A. G., Choi, J., Rha, C., St ubbe, J., and Sinskey, A. J. (2005) In vitro analysis of the chain termination react ion in the synthesis of poly-(R)-betahydroxybutyrate by the class III synthase from Allochromatium vinosum. Biomacromolecules 6, 2113-9. (69) JAGUAR (2000) Schrdinger, LLC, Portland, OR. (70) MAESTRO (2002) Schrdinger, LLC, Portland, OR. (71) Hiskey, R. G., and Jun g, J. M. (1963) Azomethane chemistry. II. Formation of peptides from oxazolidine-5-ones. J Am Chem Soc 85, 578-582. (72) Merkler, D. J., Asser, A. S., Baumgart, L. E., Carpenter, S. E., Chew, G. H., Consalvo, A. P., DeBlassio, J. L., Galloway, L. C., Lowe, A. B., Lowe, E. W. J., Lawrence King III, L., Kendig, R. D., Kline, P. C., Kulathila, R., Robert Malka, R., Merkler, K. A., McIntyre, N. R., Wilcox, B. J., and Owen, T. C. Hippurate, Substituted Hippurates, and Hippurate Analogs as Substrates and Inhibitors of Peptidylglycine -Hydroxylating Monooxygenase (PHM). in preparation 216

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CHAPTER 5: NMR Spectra Spectrum 5.1: benzaldehyde imino-oxy acetic acid 1 H NMR analysis (250MHz, MeODd 4 ) and 13 C NMR analysis (62.5MHz, MeODd 4 ) 218

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CHAPTER 5: NMR Spectra Spectrum 5.2: [ 2 H 2 ]-benzaldehyde imino-oxy acetic acid 1 H NMR analysis (250MHz, MeODd 4 ) and 13 C NMR analysis (62.5MHz, MeODd 4 ) 219

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CHAPTER 5: NMR Spectra Spectrum 5.3: para-nitro benzaldehyde imino-oxy acetic acid 1 H NMR analysis (250MHz, MeODd 4 ) and 13 C NMR analysis (62.5MHz, MeODd 4 ) 220

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CHAPTER 5: NMR Spectra Spectrum 5.4: para-methoxy benzaldehyde imino-oxy acetic acid 1 H NMR analysis (250MHz, MeODd 4 ) and 13 C NMR analysis (62.5MHz, MeODd 4 ) 221

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CHAPTER 5: NMR Spectra Spectrum 5.5: para-chloro benzaldehyde imino-oxy acetic acid 1 H NMR analysis (400MHz, DMSOd 6 ) and 13 C NMR analysis (100MHz, DMSOd 6 ) 222

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CHAPTER 5: NMR Spectra Spectrum 5.6: para-fluoro benzaldehyde imino-oxy acetic acid 1 H NMR analysis (400MHz, DMSOd 6 ) and 13 C NMR analysis (100MHz, DMSOd 6 ) 223

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CHAPTER 5: NMR Spectra Spectrum 5.7: para-hydroxy benzaldehyde imino-oxy acetic acid 1 H NMR analysis (400MHz, DMSOd 6 ) and 13 C NMR analysis (100MHz, DMSOd 6 ) 224

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CHAPTER 5: NMR Spectra Spectrum 5.8: acetophenone imino-oxy acetic acid 1 H NMR analysis (250MHz, MeODd 4 ) and 13 C NMR analysis (62.5MHz, MeOD d 4 ) 225

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CHAPTER 5: NMR Spectra Spectrum 5.9: [ 2 H 2 ] acetophenone imino-oxy acetic acid 13 C NMR analysis (62.5MHz, MeOD d 4 ) 226

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CHAPTER 5: NMR Spectra Spectrum 5.10: para-cyano acetophenone imino-oxy acetic acid 1 H NMR analysis (250MHz, MeODd 4 ) and 13 C NMR analysis (62.5MHz, MeOD d 4 ) 227

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CHAPTER 5: NMR Spectra Spectrum 5.11: para-nitro acetophenone imino-oxy acetic acid 1 H NMR analysis (250MHz, D 2 Od 2 ) and 13 C NMR analysis (62.5MHz, MeODd 4 ) 228

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CHAPTER 5: NMR Spectra Spectrum 5.12: para-methoxy acetophenone imino-oxy acetic acid 1 H NMR analysis (250MHz, MeODd 4 ) and 13 C NMR analysis (62.5MHz, MeODd 4 ) 229

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CHAPTER 5: NMR Spectra Spectrum 5.13: para-chloro acetophenone imino-oxy acetic acid 1 H NMR analysis (250MHz, MeODd 4 ) and 13 C NMR analysis (62.5MHz, MeODd 4 ) 230

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CHAPTER 5: NMR Spectra Spectrum 5.14: para-fluoro acetophenone imino-oxy acetic acid 1 H NMR analysis (62.5MHz, MeODd 4 ) 231

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CHAPTER 5: NMR Spectra Spectrum 5.15: para-hydroxy acetophenone imino-oxy acetic acid 1 H NMR analysis (250MHz, MeODd 4 ) and 13 C NMR analysis (62.5MHz, MeODd 4 ) 232

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CHAPTER 5: NMR Spectra Spectrum 5.16: para-amino acetophenone imino-oxy acetic acid 13 C NMR analysis (100MHz, DMSOd 6 ) 233

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CHAPTER 5: NMR Spectra Spectrum 5.17: -naphthaldehyde imino-oxy acetic acid 1 H NMR analysis (400MHz, DMSOd 6 ) and 13 C NMR analysis (100MHz, DMSOd 6 ) 234

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CHAPTER 5: NMR Spectra Spectrum 5.18: [ 2 H 2 ]-naphthaldehyde imino-oxy acetic acid 1 H NMR analysis (100MHz, DMSOd 6 ) 235

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CHAPTER 5: NMR Spectra Spectrum 5.19: para-(5-dimethylaminonapthalene-1-sulfonamido) acetophenone iminooxy acetic acid 1 H NMR analysis (400MHz, DMSOd 6 ) and 13 C NMR analysis (100MHz, DMSOd 6 ) 236

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237 Spectrum 5.20: [2H2]Acetylglycine 1H NMR analysis (400MHz, DMSOd6) and 13C NMR analysis (100MHz, DMSOd6)

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238 Spectrum 5.21: [1H2]Propionylglycine 1H NMR analysis (400MHz, DMSOd6) and 13C NMR analysis (100MHz, DMSOd6)

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239 Spectrum 5.22: [2H2]Propionylglycine 1H NMR analysis (400MHz, DMSOd6) and 13C NMR analysis (100MHz, DMSOd6)

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240 Spectrum 5.23: [1H2]Butyrylglycine 1H NMR analysis (400MHz, DMSOd6) and 13C NMR analysis (100MHz, DMSOd6)

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241 Spectrum 5.24: [2H2]Butyrylglycine 1H NMR analysis (400MHz, DMSOd6) and 13C NMR analysis (100MHz, DMSOd6)

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242 Spectrum 5.25: [1H2]Hexanoylglycine 1H NMR analysis (400MHz, DMSOd6) and 13C NMR analysis (100MHz, DMSOd6)

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243 Spectrum 5.26: [2H2]Hexanoylglycine 1H NMR analysis (400MHz, DMSOd6) and 13C NMR analysis (100MHz, DMSOd6)

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244 Spectrum 5.27: [1H2]Octanoylglycine 1H NMR analysis (400MHz, DMSOd6) and 13C NMR analysis (100MHz, DMSOd6)

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245 Spectrum 5.28: [2H2]Octanoylglycine 1H NMR analysis (400MHz, DMSOd6) and 13C NMR analysis (100MHz, DMSOd6)

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246 Spectrum 5.29: [1H2]Decanoylglycine 1H NMR analysis (400MHz, DMSOd6) and 13C NMR analysis (100MHz, DMSOd6)

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247 Spectrum 5.30: [2H2]Decanoylglycine 1H NMR analysis (400MHz, DMSOd6) and 13C NMR analysis (100MHz, DMSOd6)

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About the Author Neil Robin McIntyre receive d a Joint Advanced Major in Chemistry and Biology form St. Francis Xavier University (Anti gonish, Nova Scotia, Canada) in 2001. As an undergraduate, Neil was an active member of campus life employed as a resident assistant, teaching assistant for freshman chemistry labs and an assignment corrector. When not working, Neil volunteered for the campu s help-line service, instructed judo and a female self-defense class. His apprecia tion for clinical science developed as a research student at the Regional General Hospital after junior year in palliative care. It was here that the influence of chemistry in therapeutic strategies was introduced. During his senior year, Neil joined the lab of Dr. Manuel Aquino to studying the synthesis of novel ruthenium complexes. Hi s initial plan was to attend graduate school to study the development of inorganic ca talysts. This plan was altered, once a second semester module in bio-organic chemistr y introduced him to enzymatic catalysis. Following graduation, Neils Ph.D. research was performed at the University of South Florida, Chemistry Department under the guidance of Dr. David J. Merkler studying the mechanism of the enigmatic enzyme, peptidylglycine -amidating monooxygenase (PAM). When not in lab, Neil enjoys lifting we ights, judo, and doing handy work.


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Mechanistic studies of peptidylglycine alpha-amidating monooxygenase (PAM)
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ABSTRACT: Peptide hormones are responsible for cellular functions critical to the survival of an organism. Approximately 50% of all known peptide hormones are post-translationally modified at their C-terminus. Peptidylglycine alpha-amidating monooxygenase (PAM) is a bi-functional enzyme which catalyzes the activation of peptide pro-hormones. PAM also functionalizes long chain N-acylglycines suggesting a potential role in signaling as their respective fatty acid amides. As chain length increases for N-acylglycines so does the catalytic efficiency. This effect was probed further by primary kinetic isotope effects and molecular dynamics to better resolve the mechanism for improved catalytic function. The KIE showed a linear decrease with increasing chain length. Neither the minimal kinetic mechanism nor the maximal rate for substrate oxidation was observed to be altered by substrate hydrophobicity.It was concluded that KIE suppression was a function of 'Pre-organization' more efficient degenerate wave function overlap between C-H donor and Cu(II)-superoxo acceptor with increased chain length. Substrate activation is believed to be facilitated by a Cu(II)-superoxo complex formed at Cu[subscript]M. Benzaldehyde imino-oxy acetic acid undergoes non-enzymatic O-dealkylation to the corresponding oxime and glyoxylate products. This phenomena was further studied using QM/MM methodology using different Cu/O species to determine which best facilitated the dealkylation event. It was determined that radical recombination between a Cu(II)-oxyl and a substrate radical to form an unstable copper-alkoxide intermediate was best suited to carry out this reaction. Structure-function analysis was used to rationalize the electronic features which made a variety of diverse imino-oxy acetic acid analogues such unexpectedly good PAM substrates (10 Ms).To observe the effect oxygen insertion and placement had on substrates between N-benzoylglycine and benzaldehyde imino-oxy acetic acid structures, PAM activity was correlated with NBO/MEP calculations on selected PHM-docked structures. This work concluded that the imino-oxy acetic acid was a favored substrate for PAM because its oxime electronically is very similar to the amide present in glycine-extended analogues.
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