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N-acyltyramines as substrates for tyrosinase

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Title:
N-acyltyramines as substrates for tyrosinase enzymatic lag and dopamine precursor
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Shafer, Jacob A
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Kinetics
Enzyme
Quinone
Mushroom
Oxygen electrode
Dissertations, Academic -- Chemistry -- Masters -- USF   ( lcsh )
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non-fiction   ( marcgt )

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Abstract:
ABSTRACT: Tyrosinase is a widespread, highly studied and important enzyme involved in processes ranging from the browning of mushrooms to roles in mammalian cancer. The enzyme suffers from a noticeable lag phase while the enzyme generates all necessary cofactors from available substrates. There have not been significant studies of the effect on lag from moving through a family of substituted substrates. This thesis reports the results of one such study using a family of N-acyltyramines. The selection of N-acyltyramines was ideal because the substrates in this reaction may be related to synthesis of N-acyldopamines, which serve many important physiological functions. It was concluded that the product formed from N-acetyltyramine is 1-acetyl-2,3-dihydro-1H-indole-6,7-dione, a quinone.
Thesis:
Thesis (M.S.)--University of South Florida, 2009.
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Includes bibliographical references.
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by Jacob A. Shafer.
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Title from PDF of title page.
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N -Acyltyramines as Substrates for Tyrosinase: Enzymatic Lag and Dopamine Precursor by Jacob A. Shafer A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science Department of Chemistry College of Arts and Sciences University of South Florida Major Professor: David J. Merkler, Ph.D. G. King Farrington, Ph.D. Roman Manetsch, Ph.D. Mark L. McLaughlin, Ph.D. Date of Approval: March 31, 2009 Keywords: Kinetics, Enzyme, Qu inone, Mushroom, Oxygen Electrode Copyright 2009, Jacob A. Shafer

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Dedication To my family and friends thanks. “What do you say to taking chances What do you say to jumping off the edge Never knowing if there’s solid ground below Or hand to hold Or hell to pay What do you say What do you say” Celine Dion Written by Kara DioGuardi and David A. Stewart

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Acknowledgements I would like to thank Dr. David J. Merkler for giving me the opportunity to work and mature in his lab over the last several years. I would also like to thank my commi ttee members: G. King Farrington, Roman Manetsch and Mark L. McLaughlin (additionally for a year of work in his lab) for their suggestions and advice. I wish to express my thanks to Mi lena Ivkovic and Emma Farrell for their constant company and insights all along the way. I would like to acknowledge the rest of Merkler La b during my tenure: Neil McIntrye, Will Lowe (additionally for the co mputer modeling), Zhenming An, Shikha Mahajan and Sumit Handa.

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i Table of Contents List of Tables iii List of Figures iv List of Schemes x Abstract xi Chapter 1: Introduction 1 Tyrosinase 1 N -Acyldopamine 5 Chapter 2: Materials and Methods 8 Synthesis and Purification of N -Acyltyramines 8 Synthesis of N -Benzoyltyramine 9 Kinetic Assay of L3,4-Dihydroxyphenylanlinine 9 Kinetic Assays of N -Acyltyramines with Tyrosinase 10 Product Determination 10 Exploration of Enzymatic Lag 11 Modeling Experiments 11 Chapter 3: Results and Discussion 12 Kinetics 12 Docking Experiment 14 Product Determination 15 Enzymatic Lag 18 Conclusion 19 Works Cited 20 Appendices 25 Appendix A: NMR Spectra 26 Appendix B: Michaelis-Menten Graphs 36

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ii Appendix C: Oxygen Electrode Traces for Kinetic Parameters 39 Appendix D: Oxygen Electrode Traces for Lag Determination 57

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iii List of Tables Table 1: Kinetic constants for N -acyltyramines 13 Table 2: Kinetic constants and calcu lated free energies of binding for N -acyltyramines 14

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iv List of Figures Figure 1: Structure of tyrosinase 2 Figure 2: Active site of tyrosinase 3 Figure 3: Strucutres of tyramine, tyrosine and similar compounds 7 Figure 4: Processed oxygen electrode data for 50 mM tyramine with .05 mg tyrosinase 12 Figure 5: Michaelis-Menten plot of ty rosinase activity with tyramine 13 Figure 6: Active site of tyrosinase with N -acetyltyramine bound 14 Figure 7: Product structure: 1-acety l-2,3-dihydro-1H-indole-6,7-dione with 1H 16 predictions Figure 8: 1H NMR spectrum in MeOD-d4of tyrosinase reaction product where 16 N -acetyltyramine was the substrate Figure 9: Product structure: 1-acety l-2,3-dihydro-1H-indole-6,7-dione with 13C 17 predictions Figure 10: 13C NMR spectrum in MeOD-d4of tyrosinase reaction product where 17 N -acetyltyramine was the substrate Figure 11: 1H NMR spectrum in acetone-d6 of tyrosinase reaction product where 18 N -acetyltyramine was the substrate Figure 12: Lag times for tyramine and N -acyltyramines with increasing tyrosinase 19 Figure A-1: 1H NMR spectrum of N -acetyltyramine in MeOD-d4 26 Figure A-2: 13C NMR spectrum of N -acetyltyramine in MeOD-d4 27 Figure A-3: 1H NMR spectrum of N -propanoyltyramine in MeOD-d4 28 Figure A-4: 13C NMR spectrum of N -propanoyltyramine in MeOD-d4 29 Figure A-5: 1H NMR spectrum of N -butyryltyramine in MeOD-d4 30 Figure A-6: 13C NMR spectrum of N -butyryltyramine in MeOD-d4 31 Figure A-7: 1H NMR spectrum of N -pentanoyltyramine in MeOD-d4 32 Figure A-8: 13C NMR spectrum of N -pentanoyltyramine in MeOD-d4 33

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v Figure A-9: 1H NMR spectrum of N -hexanoyltyramine in MeOD-d4 34 Figure A-10: 13C NMR spectrum of N -hexanoyltyramine in MeOD-d4 35 Figure B-1: Michaelis-Menten plot of tyrosinase activity with N -acetyltyramine 36 Figure B-2: Michaelis-Menten plot of tyrosinase activity with N -propanoyltyramine 37 Figure B-3: Michaelis-Menten plot of tyrosinase activity with N -butyryltyramine 37 Figure B-4: Michaelis-Menten plot of tyrosinase activity with N -pentanoyltyramine 38 Figure B-5: Michaelis-Menten plot of tyrosinase activity with N -hexanoyltyramine 38 Figure C-1: Processed oxygen electrode data for 1 mM tyramine with 39 .05 mg tyrosinase Figure C-2: Processed oxygen electrode data for 2 mM tyramine with 40 .05 mg tyrosinase Figure C-3: Processed oxygen electrode data for 5 mM tyramine with 40 .05 mg tyrosinase Figure C-4: Processed oxygen electrode data for 15 mM tyramine with 41 .05 mg tyrosinase Figure C-5: Processed oxygen electrode data for 25 mM tyramine with 41 .05 mg tyrosinase Figure C-6: Processed oxygen electrode data for .15 mM N -acetyltyramine with 42 .025 mg tyrosinase Figure C-7: Processed oxygen electrode data for .31 mM N -acetyltyramine with 42 .025 mg tyrosinase Figure C-8: Processed oxygen electrode data for .625 mM N -acetyltyramine with 43 .025 mg tyrosinase Figure C-9: Processed oxygen electrode data for 1.25 mM N -acetyltyramine with 43 .025 mg tyrosinase Figure C-10: Processed oxygen electrode data for 2.5 mM N -acetyltyramine with 44 .025 mg tyrosinase Figure C-11: Processed oxygen electrode data for 5 mM N -acetyltyramine with 44 .025 mg tyrosinase

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vi Figure C-12: Processed oxygen electrode data for 10 mM N -acetyltyramine with 45 .025 mg tyrosinase Figure C-13: Processed oxygen electrode data for .31 mM N -propanoyltyramine with 45 .025 mg tyrosinase Figure C-14: Processed oxygen electrode data for .625 mM N -propanoyltyramine with 46 .025 mg tyrosinase Figure C-15: Processed oxygen electrode data for 1.25 mM N -propanoyltyramine with 46 .025 mg tyrosinase Figure C-16: Processed oxygen electrode data for 2.5 mM N -propanoyltyramine with 47 .025 mg tyrosinase Figure C-17: Processed oxygen electrode data for 5 mM N -propanoyltyramine with 47 .025 mg tyrosinase Figure C-18: Processed oxygen electrode data for 10 mM N -propanoyltyramine with 48 .025 mg tyrosinase Figure C-19: Processed oxygen electrode data for .31 mM N -butyryltyramine with 48 .025 mg tyrosinase Figure C-20: Processed oxygen electrode data for .625 mM N -butyryltyramine with 49 .025 mg tyrosinase Figure C-21: Processed oxygen electrode data for 1.25 mM N -butyryltyramine with 49 .025 mg tyrosinase Figure C-22.: Processed oxygen electrode data for 2.5 mM N -butyryltyramine with 50 .025 mg tyrosinase Figure C-23: Processed oxygen electrode data for 5 mM N -butyryltyramine with 50 .025 mg tyrosinase Figure C-24: Processed oxygen electrode data for 10 mM N -butyryltyramine with 51 .025 mg tyrosinase Figure C-25: Processed oxygen electrode data for .15 mM N -pentanoyltyramine with 51 .025 mg tyrosinase Figure C-26: Processed oxygen electrode data for .31 mM N -pentanoyltyramine with 52 .025 mg tyrosinase

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vii Figure C-27: Processed oxygen electrode data for .625 mM N -pentanoyltyramine with 52 .025 mg tyrosinase Figure C-28 Processed oxygen electrode data for 1.25 mM N -pentanoyltyramine with 53 .025 mg tyrosinase Figure C-29: Processed oxygen electrode data for 2 mM N -pentanoyltyramine with 53 .025 mg tyrosinase Figure C-30: Processed oxygen electrode data for .15 mM N -hexanoyltyramine with 54 .025 mg tyrosinase Figure C-31: Processed oxygen electrode data for .31 mM N -hexanoyltyramine with 54 .025 mg tyrosinase Figure C-32: Processed oxygen electrode data for .625 mM N -hexanoyltyramine with 55 .025 mg tyrosinase Figure C-33: Processed oxygen electrode data for 1.25 mM N -hexanoyltyramine with 55 .025 mg tyrosinase Figure C-34: Processed oxygen electrode data for 1.75 mM N -hexanoyltyramine with 56 .025 mg tyrosinase Figure C-35: Processed oxygen electrode data for 2.5 mM N -hexanoyltyramine with 56 .025 mg tyrosinase Figure D-1: Processed oxyge n electrode data for 6.6 mM tyramine with .025 mg 57 tyrosinase Figure D-2: Processed oxyge n electrode data for 6.6 mM tyramine with .05 mg 58 tyrosinase Figure D-3: Processed oxyge n electrode data for 6.6 mM tyramine with .075 mg 58 tyrosinase Figure D-4: Processed oxygen electrode data for 6.6 mM tyramine with 59 .1 mg tyrosinase Figure D-5 Processed oxygen electrode data for 6.6 mM tyramine with 59 .15 mg tyrosinase Figure D-6: Processed oxygen electrode data for 6.6 mM tyramine with 60 .2 mg tyrosinase

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viii Figure D-7: Processed oxygen electrode data for 1.2 mM N -acetyltyramine with 60 .00625 mg tyrosinase Figure D-8: Processed oxygen electrode data for 1.2 mM N -acetyltyramine with 61 .0125 mg tyrosinase Figure D-9: Processed oxygen electrode data for 1.2 mM N -acetyltyramine with 61 .025 mg tyrosinase Figure D-10: Processed oxygen electrode data for 1.2 mM N -acetyltyramine with 62 .05 mg tyrosinase Figure D-11: Processed oxygen electrode data for 1.2 mM N -acetyltyramine with 62 .075 mg tyrosinase Figure D-12: Processed oxygen electrode data for 1.2 mM N -acetyltyramine with 63 .1 mg tyrosinase Figure D-13: Processed oxygen electrode data for 1.3 mM N -propanoyltyramine with 63 .00625 mg tyrosinase Figure D-14: Processed oxygen electrode data for 1.3 mM N -propanoyltyramine with 64 .0125 mg tyrosinase Figure D-15: Processed oxygen electrode data for 1.3 mM N -propanoyltyramine with 64 .025 mg tyrosinase Figure D-16: Processed oxygen electrode data for 1.3 mM N -propanoyltyramine with 65 .05 mg tyrosinase Figure D-17: Processed oxygen electrode data for 1.3 mM N -propanoyltyramine with 65 .075 mg tyrosinase Figure D-18: Processed oxygen electrode data for 1.3 mM N -propanoyltyramine with 66 .1 mg tyrosinase Figure D-19: Processed oxygen electrode data for 2.3 mM N -butyryltyramine with 66 .0031 mg tyrosinase Figure D-20: Processed oxygen electrode data for 2.3 mM N -butyryltyramine with 67 .0062 mg tyrosinase Figure D-21: Processed oxygen electrode data for 2.3 mM N -butyryltyramine with 67 .0125 mg tyrosinase

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ix Figure D-22: Processed oxygen electrode data for 2.3 mM N -butyryltyramine with 68 .05 mg tyrosinase Figure D-23: Processed oxygen electrode data for 2.3 mM N -butyryltyramine with 68 .075 mg tyrosinase Figure D-24: Processed oxygen electrode data for 2.3 mM N -butyryltyramine with 69 .1 mg tyrosinase

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x List of Schemes Scheme 1: General reaction catalyzed by tyrosinase 1 Scheme 2: Proposed tyrosinase mechanism 4 Scheme 3: Proposed biosynthetic pathways for N -ayldopamine 6 Scheme 4: Synthesis of N -acyltyramines 9

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xi N -Acyltyramines as Substrates for Tyrosinase: Enzymatic Lag and Dopamine Precursor Jacob A. Shafer ABSTRACT Tyrosinase is a widespread, highly studied and important enzyme involved in processes ranging from the browning of mushrooms to roles in mammalian cancer. The enzyme suffers from a noticeable lag phase while th e enzyme generates all necessary cofactors from available substrates. There have not been significant studies of the effect on lag from moving through a family of substituted subs trates. This thesis reports the results of one such study using a family of N -acyltyramines. The selection of N -acyltyramines was ideal because the substrates in this reaction may be related to synthesis of N -acyldopamines, which serve many important physiological functions. It was concluded that the product formed from N -acetyltyramine is 1-acetyl2,3-dihydro-1H-indole-6,7-dione, a quinone.

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1 Chapter 1 Introduction Tyrosinase Tyrosinase (E.C. 1.14.18.1) is an enzyme whic h is distributed wide ly throughout all life forms [1]. The primary biological role is to produce melanin, which causes a darkening of tissues [2]. Due to its presence in mush rooms and plants, tyrosinase has been studied from the mid 1800s, as the related browning of harvested mushrooms was, and remains to be, a chief agricultural concern [3, 4]. In mammals, it was thought to be involved in cancer [5]. More recently, there has been in terest from the cosmetic industry for finding inhibitors to act as skin whiteners [6] and it is being explored for its role in many types of albinism [7]. Tyrosinase has been consider ed as a tumor suppressor; however, it might have a role in mutagenicity as a free radical producer [8, 9]. One of the primary products of tyrosinase is melanin. This is a family of pigments found in life ranging from higher mammals down to insets and arthropods. Its role in higher organisms relates to coloration, such as hair and skin, as well as protection for solar UV radiation [10, 11]. In lower organisms, the role served by tyrosi nase is more expansive, ranging from exoskeleton coloration to immune system response and wound healing [12, 13]. Scheme 1: General reaction ca talyzed by tyrosinase. R H.

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2 The enzyme is a heterotetramer, with two heavy and two light chains for a combined molecular mass of 120 kDa (Figure 1) [14]. The central domain, containing the active site, is well conserved across the various organisms [15]. Th e active site is composed of six histidines binding two copper ions, and ha s binding pockets for both the substrate and a reductant (Figure 2). Figure 1. Structure of tyrosinase. Coloring scheme: Cu (brown), O2 (red), NO3 2(blue, red) with no bound substrate [16]. The enzyme’s primary mechanism occurs in two steps using molecular oxygen both to hydroxylate monophenols to o -diphenols and to oxidize diphenols to o -quinones (Scheme 1) [10, 11]. Tyrosinase expr esses stereospecificity with regard to binding affinity, favoring D-isomers, but does not show any speci ficity towards the reaction rate [17]. There is ongoing research as to the exact mechanism used by tyrosinase. Scheme 2 shows one proposal where substrates are M, a monophenol, and D, an o -diphenol, and the active site states are Eoxoxy tyrosinase, Emmet tyrosinase and Eddeoxy tryosinase. In Eox, there is O2 bound to the Cu ions and both are in +2 oxidation state, while in Ed there is no oxygen moiety bound and the Cu ions are both in the +1 oxidation state, and the Em has a hydroxyl bound and both Cu ions are in +2 oxidation state. In this proposal, Eox can react

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3 with either D or M. In the case of M, D is formed which can be released leaving the active site as Em or the final product, a quinone, can be evolved leaving tyrosinase as Ed. If Eox reacts with D, then the quinone is made and tyrosinase ends in Em. Em binding with M is a dead-end because the hydroxyl is unable to form a new C-O bond. In order to have meaningful forward movement of this reaction scheme, the enzyme needs to be available in the Eox state or the substrate must be a diphenol. In most cases, however, the substrate is a monophenol and tyrosina se is predominately found in the Em state [18-26]. Figure 2. Active site of tyrosina se. Color scheme: Cu (orange), O2 (red) and hisitidines with no bound substrate (generated from [16]). These two factors set-up wh at is known as the lag ( ) phase of the reaction. During this time, a small amount of naturally occurring Eox enzyme (2-30%) is converting M to D where the reaction can move forward read ily. This unusual phenomenon has been studied to some extent and it is known that increasing the enzyme concentration will reduce due to the increased amount of enzyme present in Eox. Additionally, by increasing the substrate (M) concentration, will increase. This is because k-2 is larger than k3 (Scheme 2) and Em is generated along with D, leaving enzyme inactive until enough D is accumulated to make k3 favorable, and M out competes D at the active site of remaining Eox [18, 20, 22, 23].

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4 Scheme 2. Proposed tyrosinase mechanism. Em met tyrosinase, Eox oxy tyrosinase, Ed deoxy tryosinase, M monophenol, D o -diphenol, and the final product of a quinone (adapted from [21]).

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5 N-Acyldopamine Dopamines have many important biological role s. Dopamine is most widely known for its role as a neurotransmitter. Also it has a primary role as a precursor in the formation of melanin, which is involved in pigmentati on in many species and sclerotization, the hardening and coloring of exoskeletons, in ins ects [24, 26-28]. More recently the role of N -acyldopamine in brain and central ne rvous system has been explored. N Arachidonoyl-, N -palmitoyl-, N -oleoyland N -stearoyldopamine are all known to present in mammals. In the case of N -arachidonoyland N -oleoyldopamine, they bind to TRPV1 and CB1 receptors which experimentally cause hyperalgesia. Also, N -oleoyldopamine has been shown to reduce risk of ischem ia and reperfusion injury by action on the TRPV1 receptor in the heart [29]. N -Palmitoyland N -stearoyldopmaine have an unknown biological role [ 30-33]. There is also interest in N-acyldopamines as drugs for both their physical and inhibitory properties. N -Acyldopamines has been shown to selfaggregate into micelle supramolecules and th en hydrolyze slowly, gradually releasing dopamine to receptors [35]. In rats, there is evidence that N -acetyldopamine inhibits malondialdehyde production, which is tied to oxidative stress, bette r than the natural agent, melatonin[27, 36]. N -Acetyledopamine has been shown to decrease superoxide production in THP-1 derived human monocytes [38].It has been discovered that N linoleoyldopamine is produced in plants and ac ts as a strong inhib itor to arachidonate 5lipoxygenase, a good drug target in humans for asthma [37]. N -Acyldopamine biosynthesis is not co mpletely understood, and two competing pathways have been proposed (Scheme 3). Ther e is evidence that tyrosine can be directly acylated via the enzyme N acyltyrosine synthase followe d by oxidation by tyrosinase to N -acyldopamine (pathway A). An analogous pathway involves acylation of tyramine by an unknown enzyme. Alternatively, acyl groups may be transferred to tyramine or tyrosine via acyl-coenzyme A; the resulting species would then be oxidized by tyrosinase (pathway B) [32, 39, 40].

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6 Tyrosine (Figure 3) has been explored to so me extent in the literature, mostly as the primary substrate for tyro sinase with a reported KM of 1.25 mM [41]. It has been shown that tyrosinase can ca talyze the oxidation of N -acetyltyrosine to the corresponding quinone [42]. Additionally, the si milar family of compounds of N -acetyl-4-Scysteaminylphenol (Figure 3) and N -propanoyl-4-S-cysteaminyl phenol have been shown to be substrates for tyrosinase [43]. Ther e has also been some work suggesting that N methyl-, N -methoxyand N -formyltyrosine as well as the methyl ester (Figure 3) are substrates for tyrosinase [ 19, 44-46]. On the other hand, N -acyltyramines (Figure 3) are not widely studied. They have been found in trace amounts throughout the central nervous system of insects and mammals [47, 48] In honeybees, there is evidence that the ratio of tyramine and N -acetyltyramine is dependent on the presence of a queen – causing workers to convert to queens as needed. N -acetyltyramine may be a viable drug in the conversion of resistant state to sensitive state leukemia [49]. Scheme 3: Proposed bios ynthetic pathways for N -acyldopamine. A represents an acyl group. Pathway A is on the left, Pathway B is on the right.

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7 NH HO O HO O N-acetyltyrosineNH HO O HO O N-methoxytyrosineHN HO O OH N-methyltyrosineNH HO O HO O N-formyltyrosineNH2HO O O MethyltyrosineesterNH2S HO O HO 4-S-cysteaminylphenol Figure 3: Structures of tyramine, tyrosine and similar compounds. Even though most of the literature indicates that the precursors for N -acyldopamines are N -actyltyrosines, the recent discovery by a Russian group [39] that N -acyltyramines are preferred to N -acytyrosines in vivo drew our attention. As mentioned above, N acyltyramines are found in tissues where N -acyldopamines are located, but it is not known what, if any, role they serv e there. In order to further investigate the possibility of N -acyltyramines acting as precursors to N -acyldopamines, a series of N -acyltyramines, with varying acyl chain lengths, were synthesized and evaluated as tyrosinase substrates. This also gave us the opportunity to study the effect acy l chain length has on the enzymatic lag tyrosinase exhibits. Ideally, the data will show that N -acyltyramines have superior kinetic parameters to tyramine. Additionally, characterization should indicate the product to be N -acyldopamine or its derivatives.

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8 Chapter 2 Materials and Methods Mushroom Tyrosinase Mushroom tyrosinase (EC 1.14.18.1) was acquire d from Sigma and used without further purification. The enzyme was dissolv ed in 100% water and stored at 5 oC. Initially, the concentration of protein in solution was determined by Bradford assay using bovine serum albumin as a standard [50]. A base-lin e rate was established as described later in the Material and Methods chapte r under “Kinetic Assays of N -Acyltyramines Substrate Activity with Tyrosinase” where there was no EtOH added and 2 mM tyramine was used. Then to account for change in enzyme activity from day-to-day, and to account for the effect of 20% EtOH, a standa rd reaction was performed each day in the same manner as the baseline where tyramine was always 2 mM, this stock was dissolved in 100% water. The average of two correction activities was compared to the standards and a correction was multiplied into each reaction thereafter. Synthesis and Purification of N-Acyltyramines All substrates were synthesized using a m odified approach published by Johnson et al (Scheme 4) [51, 52]. In general, tyramine wa s dissolved in acetonitr ile and triethylamine, to which a 1:1 ratio of acylchloride was a dded drop wise, while vigorously stirred at room temperature. After addition, the soluti on was allowed to stir for another 90 minutes before being evaporated under a stream of air. Purification was accomplished by dissolving the product in methanol and runni ng on a silica gel column in a gradient solvent system starting at 1% methanol in dichloromethane and progressing to 20%

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9 methanol. The final spot to elute, as ch ecked on TLC run in dichlormethane:methanol (4:1) and visualized under UV light, was the desi red product. Yields for each are below: N -acetyltyramine – 40% N -propionyltyramine – 45% N -butyryltyramine – 80% N -pentanoyltyramine – 50% N -hexanoyltyramine – 50% N -Octanoyltyramine and N -decanoyltyramine were also synthesized, but due to extremely low water solubility (<< 1 nM in 20% EtOH), enzymatic activity assays were not possible, and these were not further characterized. Identity of synthesized substrates was confirmed and purity assessed by 1H and 13C NMR on iNOVA 400 MHz and processed on MestReNova (Appendix A). Scheme 4. Synthesis of N -acyltyramines. R=CH3, C2H5, C3H7, C4H9, C5H11, C7H15, C9H19, Ph. Synthesis of N-benzoyltyramine Synthesis was identical to the N -acyltyramines. It was not possible to find a solvent which would dissolve the crude product and allow for enzymatic assays; this was not characterized.

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10 Kinetic Assay of L-3,4-Dihydroxyphenylalanine The reactions were conducted on a Yellow Sp rings Instrument Model 53 oxygen monitor which is outfitted to digitally output the O2 reading into Excel ev ery second. The oxygen electrode chamber was always prepared in the following order after cleaning and the positioning of the stir bar: water, phosphate buffer, ethanol and substrate. The solution was capped with the oxygen elec trode and allowed to stir while coming to equilibrium with the water bath at 37 oC. Oxygen bubbles were aggregated and removed at least once and until there were no noticeable bubbles left. Reaction conditions were 100 mM phosphate, pH=7.0, 0.025 mg/mL tyrosinase, and th e indicated amount of substrate with a final reaction volume of 2 mL Background rates were determined with all components except enzyme present for 30 seconds followed by addition of tyrosinase over less than 10 seconds. Due to the small change in volum e, data was not collect ed during this small window. The reaction was then followed for two minutes. All calculated slopes from Excel (Figure 4) were corrected for the am ount of enzyme (0.05 mg for tyramine and 0.025 mg for other substrates ) and converted from %O2 to mol/min/mg. SigmaPlot was used to fit a Michaelis-Mente n curve (Figure 5, Appendix B). Kinetic Assays of N-Acyltyramines Substrate Activity with Tyrosinase The general reaction conditions are identical to the L-DOPA assays with the following modifications: 20% ethanol, 100 mM phosphate, pH=7.0, 0.0125-0.025 mg/mL tyrosinase (all N -acyltyramines were dissolved in 100% EtOH and were conducted at 0.0125 mg/mL tyrosinase, tyramine was dissolv ed in 100% water and was conducted at 0.025 mg/mL tyrosinase) and the indicated amou nt of substrate with a final reaction volume of 2 mL (Appendix C). Attempts were made to take substrate concentration from .1 to 10 times than KM, however, solubility constraints on N -pentanoyltyramine and N hexanoyltyramine made this impossible.

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11 Product Determination A reaction mixture was allowed to react for 24 hours at room temp erature containing 100 mM phosphate, pH=7.0, 0.05 mg/mL ty rosinase, 20% ethanol and ~500mM N acetyltyramine. The reaction was dried under a stream of air and the resulting brown solid vortexed exhaustively in 100% MeOD-d4. 1H and 13C NMR were done on iNOVA 400 MHz and processed on MestReNova (Figur e 8, 10). The sample was dried under air and dissolved in 100% acetone-d6. 1H NMR was done on iNOVA 400 MHz and processed on MestReNova (Figure 11). Exploration of Enzymatic Lag Reactions were carried out as a bove in “Kinetic Assay of L-3,4dihydroxyphenylalanine.” The substrate level was held at KM for each substrate and enzyme concentration was increased to the indicated amount (Appendix D). Modeling Experiments The crystal structure of mushroom tyrosinase (PDB ID 1WX2 [16] ) was used for grid based ligand docking. All co-crystallized ligands deemed superfluous for enzyme function were removed from the crystal stru cture and polar hydroge ns were added using AutoDockTools. Charges were then corrected for the requisite copper ions. The receptor grid was prepared with a grid point spacing of 0.2 using AutoGrid. The substrates of interest were then prepared using AutoDockT ools to define torsions, rotamers and polar hydrogens. The ligands were then docked into the active site of mushroom tyrosinase using AutoDock 4.0 [53, 54]. All default settings were utilized with the exception of increasing the number of energy evaluations from 2.5 x 104 to 2.5 x 107.

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12 Chapter 3 Results and Discussion Kinetic Evaluation of LDOPA and N-Acyltyramines Results for the kinetic constants for the six compounds studied are shown in Table 1. To verify the method, this process was done w ith the substrate L-DOPA, which is well studied in the literature. Figure 4: Processed oxygen electrode data for 50 mM tyramine with .05 mg tyrosinase. Best-fit line formulas are shown which form the basis for determining reaction and background rates. y = 0.0008x + 73.406 y = -0.4251x + 102.31 y = -0.0005x + 78.284 y = -0.3919x + 107.30 20 40 60 80 100 020406080100120140160180200%O2Time (s)50 mM Tyramine .05 mg Tyrosinase

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13 [Tyramine] (mM) 0102030405060Rate (mol/min/mg) 0 2 4 6 8 10 12 Figure 5. Michaelis-Menten plot of tyrosinase activity with tyramine. Table 1. Kinetic constants for N -acyltyramines. L-DOPA (C9H11NO4), was used as a validation, table values are experimental but match literature values [19, 55]. Substrate KM (mM) Vmax (mol/min/mg) Relative Vmax/KM L-DOPA 1.4 .38 140 12 100 Tyramine 6.6 .88 12 .48 1 N -acetyltyramine 1.2 .27 37 2.6 16.4 N -propanoyltyramine 1.3 .13 19 .63 7.7 N -butyryltyramine 2.3 .20 36 1.2 8.2 N -pentanoyltyramine 1.7 .28 19 1.8 5.9 N -hexanoyltyramine 1.9 .30 31 2.7 8.6 Results show that N -acyltyramines are stubstrates for tyrosinase. It is clear that all the N acyltyramines had higher binding af finity and turnover values than tyramine. There is little variation in Vmax/KM values; however, N -acetyltyramine exhibited the highest turnover and tightest binding valu es of the substituted tyramine s. The acyl chain length does not appear to have an effect on the binding properties, as all KMs are in the same range.

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14 Docking Experiments Tyramine and N -acelytyramines with chain lengths between two and four carbons were docked into the mushroom tyrosinase crystal structure using AutoDock 4.0 (Figure 6) in order to evaluate their relative binding energi es (Table 2). The logarithmic nature of calculated binding energies indicates all KM values should be of the same magnitude, which is in agreement with experimental values. Table 2. Kinetic constants and calcul ated free energy of binding for N -acyltyramines. Substrate KM (mM) Calculated Free Energy of Binding (kcal/mol) Tyramine 6.6 .9 -4.55 -2.5 N -acetyltyramine 1.2 .3 -4.28 -2.5 N -propanoyltyramine 1.3 .1 -4.43 -2.5 N -butyryltyramine 2.3 .2 -4.29 -2.5 N -pentanoyltyramine 1.7 .3 -4.37 -2.5 N -hexanoyltyramine 1.9 .3 -4.72 -2.5 Figure 6. Active site of tyrosinase with N -acetyltyramine bound. Co lor scheme: Cu (orange), O2 (red), hisitidines and bound N -acetyltyramine (generated from [16]).

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15 Product Determination A reaction was conducted as described in “Pro duct Determination” in the Materials and Methods section, where the solution was a dark brown color at the end. The sample was dried under air leaving a br own solid which consisted of the product, unreacted N acetyltyramine, enzyme and buffer. Only N -acetyltyramine and product would be expected to dissolve in MeOH; the solid was exhaustively vortexed in MeOD-d4 in 1H and 13C NMR taken on iNOVA 400 and processed w ith MestReNova (Figure 8, 10). The NMR appears to be of a pure compound and th e peaks can be assigned to the expected product as indicated for 1H (Figure 7, 8), and 13C (Figure 9, 10). Note that in MeOD-d4, there is a pentet overlapping the expected triple t at 3.3 ppm, which makes integration unsuccessful. Based on the 1H and 13C spectra, it was determined that the product was 1acetyl-2,3-dihydro-1H-indole-6,7-dione. In or der to verify that the product was a quinone rather than a diphenol, the sample wa s dried as before and dissolved in acetoned6; NMR was run on an iNOVA 400 and processe d with MestReNova (Figure 11). The resulting spectrum showed no evidence of an OH band suggesting that the compound is a quinone. This is not an unreasonable outcome as Borovanksy et al proposed cyclization after N -acetyldopamine is oxidized [28].

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16 Figure 7. Product structure: 1-acetyl-2,3-dihydro-1H-indole-6,7-dione with 1H predictions. Figure 8. 1H NMR spectrum in MeOD-d4 of tyrosinase reaction product where N -acetyltyramine was the substrate. a b c d e a b c d e

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17 N O O O Figure 9. Product structure: 1-acetyl-2,3-dihydro-1H-indole-6,7-dione with 13C predictions. Figure 10. 13C NMR spectrum in MeOD-d4 of ryrosinase reaction product where N -acetyltyramine was the substrate in MeOD-d4. a a b c d e f g h i j b c d e f g h i j

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18 Figure 11. 1H NMR spectrum in acetone-d6 of tyrosinase reaction product where N -acetyltyramine was the substrate in acetone-d6. Enzymatic Lag The reaction was conducted as described above in “Exploration of Enzymatic Lag” in Materials and Methods section as well as “Kinetic Assays.” Each substrate was held at KM and tyrosinase concentration was incr eased. This was not feasible with N pentanoyltyramine and N -hexanoyltyramine because their KM was roughly at the limit of solubility. The oxygen electrode data is in Appendix D. A graph of the lag times is shown in Figure 12. Lag decreases as enzyme concentration increases, in agreement with the literature. This is due to the low natural abundance of tyrosinase in the Eox state increasing. Lag is shorter for acyltyramines over tyramine, but is unaffected by the change in chain length. The decrease in lag is likely caused by the larger Vmax/KM

PAGE 33

19 leading to faster accumulation of di phenol which can then generate Eox,the active form of the enzyme. Figure 12. Lag times for tyramine and N -acyltyramines with increasing tyrosinase. Conclusion N -acyltyramines were shown to be substrates for tyrosinase, suggesting that they could act as the precursors to N -acyldopamines. Furthermore, N -acyltyramines have substantially higher Vmax/KM than tyramine; even though, changing the length of the substituent does not have a significant effect on KM or Vmax/KM values. While is shorter for N -acyltyramines than for tyramine, changing chain lengths has no effect. The observed decrease in lag can be attributed to higher Vmax/KM values for N -acyltyramines than tyramine. The product is exactly what is expected after formation of N acyldopamine, which further supports N -acyltyramines as a precursor for N acyldopamines. In order to get a more definitive answer, a study of N -acyltyrosines as precursors for N -acyldopamines should be conducted. 0 20 40 60 80 100 120 140 160 00.050.10.150.20.25Lag Time (sec)[Tyrosinase](mg)Tyrosinase Lag Tyramine N acetyltyramine N propanoyltyramine N butyltyramine

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20 Works Cited 1. van Gelder, C.W., W.H. Fl urkey, and H.J. Wichers, Sequence and structural features of plant and fungal tyrosinases. Phytochemistry, 1997. 45 (7): p. 1309-23. 2. Prota, G., Progress in the chemistry of me lanins and related metabolites. Med. Res. Rev., 1988. 8 (4): p. 525-56. 3. Bourquelot, E. and A. Bertrand, A re-examination of the Raper's scheme: Cyclodopa as a biological precursor of eumelanin. C. R. Soc. Biol., 1895. 47 : p. 582-584. 4. Martinez, M.V. and J.R. Whitaker, The biochemsitry and control of enzymatic browning. Trends Food Sci. Technol., 1995. 6 : p. 195-200. 5. Nishioka, K., Particulate tyrosinase of human malignant melanoma. Solubilization, purification following tr ypsin treatment, and characterization. Eur. J. Biochem., 1978. 85 (1): p. 137-46. 6. Yagi, A., T. Kanbara, and N. Morinobu, Inhibition of mushroom-tyrosinase by aloe extract. Planta. Med., 1987. 53 (6): p. 515-7. 7. Ray, K., M. Chaki, and M. Sengupta, Tyrosinase and ocular di seases: some novel thoughts on the molecular basis of oc ulocutaneous albinism type 1. Prog. Retin. Eye Res., 2007. 26 (4): p. 323-58. 8. Vogel, F.S., et al., gamma-L-Glutaminyl-4-hydroxybenzene, an inducer of cryptobiosis in Agaricus bisporus and a source of specific metabolic inhibitors for melanogenic cells. Cancer Res., 1977. 37 (4): p. 1133-6. 9. Papaparaskeva-Petrides, C., C. Ioannides, and R. Walker, Contribution of phenolic and quinonoid structures in the mu tagenicity of the edible mushroom. Food Chem. Toxicol., 1993. 31 : p. 561-567.

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21 10. Kobayashi, T., et al., Modulation of melanogenic pr otein expression during the switch from euto pheomelanogenesis. J. Cell. Sci., 1995. 108 (Pt 6) : p. 2301-9. 11. Olivares, C., et al., The 5,6-dihydroxyindole-2-carbo xylic acid (DHICA) oxidase activity of human tyrosinase. Biochem. J., 2001. 354 (Pt 1): p. 131-9. 12. Johansson, M.W. and K. Soderhall, Cellular immunity in crustaceans and the proPO system. Parasitol. Today, 1989. 5 (6): p. 171-6. 13. Lai-Fook, J., J. Invest. Physiol., 1966. 12 : p. 195-226. 14. Strothkemp, K.G., R.L. Jolley, and H.S. Mason, Quarternary structure of mushroom tyrosinase. Biochem. Biophys. Res. Commun., 1976. 70 : p. 519-524. 15. Jackman, M.P., A. Hajnal, and K. Lerch, Albino mutants of Streptomyces glaucescens tyrosinase. Biochem. J., 1991. 274 ( Pt 3) : p. 707-13. 16. Matoba, Y., et al., Crystallographic evidence that th e dinuclear copper center of tyrosinase is flexible during catalysis. J. Biol. Chem., 2006. 281 (13): p. 8981-90. 17. Espin, J.C., et al., Study of stereospecificit y in mushroom tyrosinase. Biochem. J., 1998. 331 (Pt 2) : p. 547-51. 18. Cooksey, C.J., et al., Evidence of the indirect formation of the catecholic intermediate substrate res ponsible for the autoactivati on kinetics of tyrosinase. J. Biol. Chem., 1997. 272 (42): p. 26226-35. 19. Espin, J.C., et al., Kinetic characterization of th e substrate specificity and mechanism of mushroom tyrosinase. Eur. J. Biochem., 2000. 267 (5): p. 1270-9. 20. Fenoll, L.G., et al., Michaelis constants of mushroom tyrosinase with respect to oxygen in the presence of monophenols and diphenols. Int. J. Biochem. Cell Biol., 2002. 34 (4): p. 332-6. 21. Fenoll, L.G., et al., Kinetic characterisation of the reaction mechanism of mushroom tyrosinase on tyramine/dopam ine and L-tyrosine methyl esther/L-dopa methyl esther. Int J. Biochem. Cell Biol., 2002. 34 (12): p. 1594-1607. 22. Molina, F.G., et al., An approximate analytical so lution to the lag period of monophenolase activity of tyrosinase. Int. J. Biochem. Cell Biol., 2007. 39 (1): p. 238-52.

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22 23. Penalver, M.J., et al., Reaction mechanism to exp lain the high kinetic autoactivation of tyrosinase. J. Mol. Catal. B, 2005. 33 : p. 35-42. 24. Rodriguez-Lopez, J.N., et al., Analysis of a kinetic model for melanin biosynthesis pathway. J. Biol. Chem., 1992. 267 (6): p. 3801-10. 25. Ros-Martinez, J.R., et al., Discrimination between two kinetic mechanisms for the monophenolase activity of tyrosinase. Biochem. J., 1993. 294 ( Pt 2) : p. 621-2. 26. Sugumaran, M., Molecular mechanisms for mammalian melanogenesis. Comparison with insect cuticular sclerotization. FEBS Lett., 1991. 295 (1-3): p. 233-9. 27. Bobrov, M.Y., et al., Antioxidant and neuroprotective properties of Narachidonoyldopamine. Neurosci. Lett., 2008. 431 (1): p. 6-11. 28. Borovansky, J., et al., Mechanistic studies of melanoge nesis: the influence of Nsubstitution on dopamine quinone cyclization. Pigment. Cell Res., 2006. 19 (2): p. 170-8. 29. Zhong, B. and D.H. Wang, N-oleoyldopamine, a novel endogenous capsaicin-like lipid, protects the heart against ischemia -reperfusion injury via activation of TRPV1. Am. J. Physiol. Heart Circ. Physiol., 2008. 295 (2): p. H728-35. 30. Chu, C.J., et al., N-oleoyldopamine, a novel endoge nous capsaicin-like lipid that produces hyperalgesia. J. Biol. Chem., 2003. 278 (16): p. 13633-9. 31. De Petrocellis, L., et al., Actions of two naturally occurring saturated Nacyldopamines on transient receptor poten tial vanilloid 1 (TRPV1) channels. Br. J. Pharmacol., 2004. 143 (2): p. 251-6. 32. Huang, S.M., et al., An endogenous capsaicin-like s ubstance with high potency at recombinant and native vanilloid VR1 receptors. Proc. Natl. Acad. Sci. U S A, 2002. 99 (12): p. 8400-5. 33. Starowicz, K., S. Nigam, and V. Di Marzo, Biochemistry and pharmacology of endovanilloids. Pharmacol. Ther., 2007. 114 (1): p. 13-33. 34. Smith, G.R.E.A., New inhibitors of sepiapterin reductase. J. Biol. Chem., 1992. 267 : p. 5599-5607.

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23 35. Pokorski, M. and Z. Matysiak, Fatty acid acylation of dopamine in the carotid body. Med. Hypotheses, 1998. 50 (2): p. 131-3. 36. Oxenkrug, G.F. and P.J. Requintina, N-acetyldopamine inhibits rat brain lipid peroxidation induced by lipopolysaccharide. Ann. N Y Acad. Sci, 2005. 1053 : p. 394-9. 37. Tseng, C.F., et al., Inhibition of in vitro pr ostaglandin and leukotriene biosyntheses by cinnamoyl-beta-pheneth ylamine and N-acyldopamine derivatives. Chem. Pharm. Bull. (Tokyo), 1992. 40 (2): p. 396-400. 38. Perianayagam, M.C. and B.L. Jaber, Endotoxin-binding affinity of sevelamer hydrochloride. Am. J. Nephrol., 2008. 28 (5): p. 802-7. 39. Akimov, M.G., et al., [New aspects of biosynthesis and metabolism of Nacyldopamines in rat tissues]. Bioorg. Khim., 2007. 33 (6): p. 648-52. 40. Huang, S.M., et al., Identification of a new class of molecules, the arachidonyl amino acids, and characterization of one member that inhibits pain. J. Biol. Chem., 2001. 276 (46): p. 42639-44. 41. Ito, M. and K. Oda, An organic solvent resistant tyrosinase from Streptomyces sp. REN-21: purification and characterization. Biosci. Biotechnol. Biochem., 2000. 64 (2): p. 261-7. 42. Kahn, V. and N. Ben-Shalom, N-acetyl-L-tyrosine (NAT ) as a substrate for mushroom tyrosinase. Pigment Cell Res., 1998. 11 (1): p. 24-33. 43. Gili, A., et al., Comparison of in vitro cytotoxic ity of N-acetyl and N-propionyl derivatives of phenolic thioether amines in melanoma and neuroblastoma cells and the relationship to tyrosinase and ty rosine hydroxylase enzyme activity. Melanoma Res., 2000. 10 (1): p. 9-15. 44. Garcia-Molina, F., et al., Kinetic study of monophenol and o-diphenol binding to oxytyrosinase. J. Mol. Catal. B, 2005. 32 : p. 185-192. 45. Granata, A., et al., Mechanistic insight into the activity of tyrosinase from variable-temperature studies in an aqueous/organic solvent. Chemistry, 2006. 12 (9): p. 2504-14.

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24 46. Hara, T., et al., Purification and kinetic properti es of phenoloxidase from pupae of the housefly. Agric. Biol. Chem., 1989. 53 : p. 1387-1393. 47. Powell, P.R., et al., Analysis of biogenic amine va riability among individual fly heads with micellar electrokinetic c apillary chromatography-electrochemical detection. Anal. Chem., 2005. 77 (21): p. 6902-8. 48. Macfarlane, R.G., et al., Identification and quantitation of N-acetyl metabolites of biogenic amines in the thoracic nervous system of the locust, Schistocerca gregaria, by gas chromatography-negative-ion chemical ionisation mass spectrometry. J. Chromatogr., 1990. 532 (1): p. 13-25. 49. Kunimoto, S., et al., Reversal of resistance by N-a cetyltyramine or N-acetyl-2phenylethylamine in doxorubicinresistant leukemia P388 cells. J. Antibiot. (Tokyo), 1987. 40 (11): p. 1651-2. 50. Bradford, M.M., A rapid and sensitive method fo r the quantitation of microgram quantities of protein utilizing the principle of prot ein-dye binding. Anal. Biochem., 1976. 72 : p. 248-54. 51. Jonsson, K.O., et al., Effects of homologues and analogues of palmitoylethanolamide upon the i nactivation of th e endocannabinoid anandamide. Br. J. Pharmacol., 2001. 133 (8): p. 1263-75. 52. Lambert, D.M., et al., Analogues and homologues of N-palmitoylethanolamide, a putative endogenous CB(2) cannabinoid, as potential ligands fo r the cannabinoid receptors. Biochim. Biophys. Acta, 1999. 1440 (2-3): p. 266-74. 53. Huey, R., et al., A semiempirical free energy fo rce field with charge-based desolvation. J. Comput. Chem., 2007. 28 (6): p. 1145-52. 54. Morris, G.M., et al., Automated docking using a La marckian genetic algorithm and empirical binding free energy funtion. J. Comput. Chem., 1998. 19 : p. 16391662. 55. Espin, J.C., S. Jolivet, and H.J. Wichers, Kinetic study of the oxidation of gammaL-glutaminyl-4-hydroxybenzene catalyze d by mushroom (Agaricus bisporus) tyrosinase. J Agric. Food Chem., 1999. 47 (9): p. 3495-502.

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25 Appendices

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Appendix A: NMR Spectra Figure A-1. 1H NMR spectrum of N -acetyltyramine in MeOD-d4. 26

PAGE 41

Figure A-2. 13C NMR spectrum of N -acetyltyramine in MeOD-d4. 27

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Figure A-3. 1H NMR spectrum of N -propanoyltyramine in MeOD-d4. 28

PAGE 43

Figure A-4. 13C NMR spectrum of N -propanoyltyramine in MeOD-d4. 29

PAGE 44

Figure A-5. 1H NMR spectrum of N -butyryltyramine in MeOD-d4. 30

PAGE 45

Figure A-6. 13C NMR spectrum of N -butyryltyramine in MeOD-d4. 31

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Figure A-7. 1H NMR spectrum of N -pentanoyltyramine in MeOD-d4. 32

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Figure A-8. 13C NMR spectrum of N -pentanoyltyramine in MeOD-d4. 33

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Figure A-9. 1H NMR spectrum of N -hexanoyltyramine in MeOD-d4. 34

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Figure A-10. 13C NMR spectrum of N -hexanoyltyramine in MeOD-d4. 35

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36 Appendix B: Michaelis-Menten Graphs [N-Acetyltyramine] (mM) 024681012Rate (mol/min/mg) 0 10 20 30 40 Figure B-1. Michaelis-Menten plot of tyrosinase activity with N -acetyltyramine.

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37 [N-Propanoyltyramine] (mM) 024681012Rate (mol/min/mg) 0 2 4 6 8 10 12 14 16 18 20 Figure B-2. Michaelis-Menten plot of tyrosinase activity with N -propanoyltyramine. [N-Butyryltyramine] (mM) 024681012Rate (mol/min/mg) 0 5 10 15 20 25 30 35 Figure B-3. Michaelis-Menten plot of tyrosinase activity with N -butyryltyramine.

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38 [N-Pentanoyltyramine] (mM) 0.00.51.01.52.02.5Rate (mol/min/mg) 0 2 4 6 8 10 12 Figure B-4. Michaelis-Menten plot of tyrosinase activity with N -pentanoyltyramine. [N-Hexanoyltyramine] (mM) 0.00.51.01.52.02.53.0Rate (mol/min/mg) 0 2 4 6 8 10 12 14 16 18 20 Figure B-5. Michaelis-Menten plot of tyrosinase activity with N -hexanoyltyramine.

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39 Appendix C: Oxygen Electrode Traces for Kinetic Parameters Figure C-1. Processed oxygen electrode data for 1 mM tyramine with .05 mg tyrosinase. Best-fit line formulas are shown which form the basis for determining reaction and background rates. y = -0.0311x + 83.329y = -0.0687x + 85.645 y = 0.0034x + 81.59 y = -0.0628x + 85.2820 20 40 60 80 100 020406080100120140160180200%O2Time (s)1 mM Tyramine .05 mg Tyrosinase

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40 Figure C-2. Processed oxygen electrode data for 2 mM tyramine with .05 mg tyrosinase. Best-fit line formulas are shown which form the basis for determining reaction and background rates. Figure C-3. Processed oxygen electrode data for 5 mM tyramine with .05 mg tyrosinase. Best-fit line formulas are shown which form the basis for determining reaction and background rates. y = 0.0051x + 94.645y = -0.1183x + 103.35 y = 0.0005x + 84.068 y = -0.0938x + 90.4140 20 40 60 80 100 020406080100120140160180200%O2Time (s)2 mM Tyramine .05 mg Tyrosinase y = 0.0042x + 82.637y = -0.1954x + 97.12 y = -0.0062x + 83.342y = -0.2238x + 99.0360 20 40 60 80 100 020406080100120140160180200%O2Time (s)5 mM Tyramine .05 mg Tyrosinase

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41 Figure C-4. Processed oxygen electro de data for 15 mM tyramine with .05 mg tyrosinase. Best-fit line formulas are shown which form the basis fo r determining reaction and background rates. Figure C-5. Processed oxygen electro de data for 25 mM tyramine with .05 mg tyrosinase. Best-fit line formulas are shown which form the basis fo r determining reaction and background rates. y = 0.0052x + 77.839y = -0.3112x + 101.13 y = -0.0084x + 83.606y = -0.2912x + 107.930 20 40 60 80 100 020406080100120140160180200%O2Time (s)15 mM Tyramine .05 mg Tyrosinase y = -0.004x + 78.126y = -0.3508x + 107.24 y = 0.0061x + 76.412y = -0.3805x + 99.4060 20 40 60 80 100 020406080100120140160180200%O2Time (s)25 mM Tyramine .05 mg Tyrosinase

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42 Figure C-6. Processed oxygen electrode data for .15 mM N -acetyltyramine with .025 mg tyrosinase. Best-fit line formulas are shown which form the basis for determining rea ction and background rates. Figure C-7. Processed oxygen electrode data for .31 mM N -acetyltyramine with .025 mg tyrosinase. Best-fit line formulas are shown which form the basis for determining rea ction and background rates. y = -0.0108x + 92.573y = -0.1057x + 97.491 y = -0.0211x + 95.999 y = -0.0888x + 100.820 20 40 60 80 100 020406080100120140160180200%O2Time (s).15 mM N-Acetyltyramine .025 mg Tyrosinase y = -0.0159x + 90.933y = -0.1672x + 102.63 y = -0.0128x + 93.62y = -0.1316x + 103.140 20 40 60 80 100 020406080100120140160180200%O2Time (s).31 mM N-Acetyltyramine .025 mg Tyrosinase

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43 Figure C-8. Processed oxygen el ectrode data for .625 mM N -acetyltyramine with .025 mg tyrosinase. Best-fit line formulas are shown which form the basis for determining rea ction and background rates. Figure C-9. Processed oxygen el ectrode data for 1.25 mM N -acetyltyramine with .025 mg tyrosinase. Best-fit line formulas are shown which form the basis for determining rea ction and background rates. y = -0.0323x + 100.19y = -0.2517x + 120.13 y = -0.0207x + 94.404 y = -0.2124x + 112.130 20 40 60 80 100 020406080100120140160180200%O2Time (s).625 mM N-Acetyltyramine .025 mg Tyrosinase y = -0.0076x + 98.999y = -0.4165x + 136.25 y = -0.0223x + 97.559y = -0.3483x + 127.090 20 40 60 80 100 020406080100120140160180200%O2Time (s)1.25 mM N-Acetyltyramine .025 mg Tyrosinase

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44 Figure C-10. Processed oxygen electrode data for 2.5 mM N -acetyltyramine with .025 mg tyrosinase. Best-fit line formulas are shown which form the basis for determining rea ction and background rates. Figure C-11. Processed oxygen electrode data for 5 mM N -acetyltyramine with .025 mg tyrosinase. Best-fit line formulas are shown which form the basis for determining rea ction and background rates. y = -0.002x + 94.885y = -0.461x + 138.75 y = -0.0004x + 94.171y = -0.5257x + 146.690 20 40 60 80 100 020406080100120140160180200%O2Time (s)2.5 mM N-Acetyltyramine .025 mg Tyrosinase y = 0.0016x + 97.898y = -0.5037x + 144.28 y = -0.0061x + 92.346y = -0.5366x + 147.80 20 40 60 80 100 020406080100120140160180200%O2Time (s)5 mM N-Acetyltyramine .025 mg Tyrosinase

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45 Figure C-12. Processed oxygen electrode data for 10 mM N -acetyltyramine with .025 mg tyrosinase. Best-fit line formulas are shown which form the basis for determining rea ction and background rates. Figure C-13. Processed oxygen electrode data for .31 mM N -propanoyltyramine with .025 mg tyrosinase. Best-fit line formul as are shown which form the ba sis for determining reaction and background rates. y = 0.0051x + 95.166y = -0.5787x + 156.44 y = 0.0017x + 97.574y = -0.5974x + 158.460 20 40 60 80 100 020406080100120140160180200%O2Time (s)10 mM N-Acetyltyramine .025 mg Tyrosinase y = -0.0026x + 85.65y = -0.1302x + 97.23 y = -0.0017x + 84.165y = -0.1284x + 95.9780 20 40 60 80 100 020406080100120140160180200%O2Time (s).31 mM N-Propanoyltyramine .025 mg Tyrosinase

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46 Figure C-14. Processed oxygen electrode data for .625 mM N -propanoyltyramine with .025 mg tyrosinase. Best-fit line formul as are shown which form the ba sis for determining reaction and background rates. Figure C-15. Processed oxygen electrode data for 1.25 mM N -propanoyltyramine with .025 mg tyrosinase. Best-fit line formul as are shown which form the ba sis for determining reaction and background rates. y = -7E-15x + 85.086y = -0.2459x + 104.48 y = -0.0141x + 88.104y = -0.2337x + 105.120 20 40 60 80 100 020406080100120140160180200%O2Time (s).625 mM N-Propanoyltyramine .025 mg Tyrosinase y = -0.0044x + 83.693y = -0.3565x + 115.83 y = -0.0045x + 84.581y = -0.3624x + 115.890 20 40 60 80 100 020406080100120140160180200%O2Time (s)1.25 mM mM N-Propanoyltyramine .025 mg Tyrosinase

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47 Figure C-16. Processed oxygen electrode data for 2.5 mM N -propanoyltyramine with .025 mg tyrosinase. Best-fit line formul as are shown which form the ba sis for determining reaction and background rates. Figure C-17. Processed oxygen electrode data for 5 mM N -propanoyltyramine with .025 mg tyrosinase. Best-fit line formul as are shown which form the ba sis for determining reaction and background rates. y = -0.0048x + 85.389y = -0.5341x + 133.05 y = 0.0075x + 85.309 y = -0.5125x + 131.280 20 40 60 80 100 020406080100120140160180200%O2Time (s)2.5 mM N-Propanoyltyramine .025 mg Tyrosinase y = -0.0031x + 91.009y = -0.6097x + 148.45 y = 9E-15x + 86.97y = -0.5553x + 147.240 20 40 60 80 100 020406080100120140160180200%O2Time (s)5 mM N-Propanoyltyramine .025 mg Tyrosinase

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48 Figure C-18. Processed oxygen electrode data for 10 mM N -propanoyltyramine with .025 mg tyrosinase. Best-fit line formul as are shown which form the ba sis for determining reaction and background rates. Figure C-19. Processed oxygen electrode data for .31 mM N -butyryltyramine with .025 mg tyrosinase. Best-fit line formul as are shown which form the ba sis for determining reaction and background rates. y = -0.0024x + 95.935y = -0.6087x + 159.34 y = -0.0025x + 93.593 y = -0.6663x + 161.440 20 40 60 80 100 020406080100120140160180200%O2Time (s)10 mM N-Propanoyltyramine .025 mg Tyrosinase y = -0.0044x + 93.843y = -0.1212x + 100.79 y = -0.0044x + 91.442 y = -0.1074x + 97.1870 20 40 60 80 100 020406080100120140160180200%O2Time (s).31 mM N-Butylyltyramine .025 mg Tyrosinase

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49 Figure C-20. Processed oxygen electrode data for .625 mM N -butyryltyramine with .025 mg tyrosinase. Best-fit line formul as are shown which form the ba sis for determining reaction and background rates. Figure C-21. Processed oxygen electrode data for 1.25 mM N -butyryltyramine with .025 mg tyrosinase. Best-fit line formul as are shown which form the ba sis for determining reaction and background rates. y = -0.0087x + 89.743y = -0.1757x + 102.38 y = -0.0022x + 89.895y = -0.1863x + 101.550 20 40 60 80 100 020406080100120140160180200%O2Time (s).625 mM N-Butylyltyramine .025 mg Tyrosinase y = 0.0029x + 92.293y = -0.2439x + 110.53 y = 0.0068x + 91.442 y = -0.3024x + 111.880 20 40 60 80 100 020406080100120140160180200%O2Time (s)1.25 mM N-Butylyltyramine .025 mg Tyrosinase

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50 Figure C-22. Processed oxygen electrode data for 2.5 mM N -butyryltyramine with .025 mg tyrosinase. Best-fit line formul as are shown which form the ba sis for determining reaction and background rates. Figure C-23. Processed oxygen electrode data for 5 mM N -butyryltyramine with .025 mg tyrosinase. Best-fit line formulas are shown which form the basis for determining rea ction and background rates. y = 0.0043x + 91.997y = -0.4066x + 122.05 y = -0.0091x + 91.991y = -0.4582x + 124.210 20 40 60 80 100 020406080100120140160180200%O2Time (s)2.5 mM N-Butylyltyramine .025 mg Tyrosinase y = -0.0098x + 94.968y = -0.5779x + 134.4 y = -0.0012x + 93.173y = -0.5984x + 134.840 20 40 60 80 100 020406080100120140160180200%O2Time (s)5 mM N-Butylyltyramine .025 mg Tyrosinase

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51 Figure C-24. Processed oxygen electrode data for 10 mM N -butyryltyramine with .025 mg tyrosinase. Best-fit line formulas are shown which form the basis for determining rea ction and background rates. Figure C-25. Processed oxygen electrode data for .15 mM N -pentanoyltyramine with .025 mg tyrosinase. Best-fit line formul as are shown which form the ba sis for determining reaction and background rates. y = -0.0019x + 89.211y = -0.6747x + 139.39 y = -0.0021x + 92.357y = -0.6509x + 133.730 20 40 60 80 100 020406080100120140160180200%O2Time (s)10 mM N-Butylyltyramine .025 mg Tyrosinase y = -0.0091x + 92.273y = -0.0572x + 94.921 y = 0.002x + 95.894y = -0.0696x + 99.5750 20 40 60 80 100 020406080100120140160180200%O2Time (s).15 mM N-Pentanoyltyramine .025 mg Tyrosinase

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52 Figure C-26. Processed oxygen electrode data for .31 mM N -pentanoyltyramine with .025 mg tyrosinase. Best-fit line formul as are shown which form the ba sis for determining reaction and background rates. Figure C-27. Processed oxygen electrode data for .625 mM N -pentanoyltyramine with .025 mg tyrosinase. Best-fit line formul as are shown which form the ba sis for determining reaction and background rates. y = -0.0007x + 94.51y = -0.1534x + 104.97 y = -0.0097x + 98.012y = -0.1239x + 106.50 20 40 60 80 100 020406080100120140160180200%O2Time (s).31 mM N-Pentanoyltyramine .025 mg Tyrosinase y = -0.0041x + 94.205y = -0.204x + 109.48 y = 0.0014x + 93.079y = -0.2057x + 108.370 20 40 60 80 100 020406080100120140160180200%O2Time (s).625 mM N-Pentanoyltyramine .025 mg Tyrosinase

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53 Figure C-28. Processed oxygen electrode data for 1.25 mM N -pentanoyltyramine with .025 mg tyrosinase. Best-fit line formul as are shown which form the ba sis for determining reaction and background rates. Figure C-29. Processed oxygen electrode data for 2 mM N -pentanoyltyramine with .025 mg tyrosinase. Best-fit line formul as are shown which form the ba sis for determining reaction and background rates. y = -0.0076x + 95.465y = -0.3146x + 124.27 y = -0.0007x + 94.679y = -0.3019x + 122.320 20 40 60 80 100 020406080100120140160180200%O2Time (s)1.25 mM N-Pentanoyltyramine .025 mg Tyrosinase y = 0.0068x + 94.636 y = -0.4062x + 130.95 y = -0.0062x + 92.82y = -0.4206x + 130.780 10 20 30 40 50 60 70 80 90 100 020406080100120140160180200%O2Time (s)2 mM N-Pentanoyltyramine .025 mg Tyrosinase

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54 Figure C-30. Processed oxygen electrode data for .15 mM N -hexanoyltyramine with .025 mg tyrosinase. Best-fit line formul as are shown which form the ba sis for determining reaction and background rates. Figure C-31. Processed oxygen electrode data for .31 mM N -hexanoyltyramine with .025 mg tyrosinase. Best-fit line formul as are shown which form the ba sis for determining reaction and background rates. y = 0.0032x + 96.693y = -0.0487x + 99.532 y = 0.0027x + 94.029y = -0.0488x + 96.720 20 40 60 80 100 020406080100120140160180200%O2Time (s).15 mM N-Hexanoyltyramine .025 mg Tyrosinase y = -0.0132x + 96.919y = -0.0869x + 102.05 y = -0.0016x + 97.641y = -0.0782x + 102.780 20 40 60 80 100 020406080100120140160180200%O2Time (s) .31 mM N-Hexanoyltyramine .025 mg Tyrosinase

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55 Figure C-32. Processed oxygen electrode data for .625 mM N -hexanoyltyramine with .025 mg tyrosinase. Best-fit line formul as are shown which form the ba sis for determining reaction and background rates. Figure C-33. Processed oxygen electrode data for 1.25 mM N -hexanoyltyramine with .025 mg tyrosinase. Best-fit line formul as are shown which form the ba sis for determining reaction and background rates. y = -0.0068x + 98.033y = -0.1334x + 106.19 y = -0.0023x + 96.664 y = -0.1204x + 104.920 20 40 60 80 100 020406080100120140160180200%O2Time (s).625 mM N-Hexanoyltyramine .025 mg Tyrosinase y = -0.0043x + 103.05y = -0.217x + 117.87 y = -0.0057x + 99.293 y = -0.2231x + 115.040 20 40 60 80 100 020406080100120140160180200%O2Time (s)1.25 mM N-Hexanoyltyramine .025 mg Tyrosinase

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56 Figure C-34. Processed oxygen electrode data for 1.75 mM N -hexanoyltyramine with .025 mg tyrosinase. Best-fit line formul as are shown which form the ba sis for determining reaction and background rates. Figure C-35. Processed oxygen electrode data for 2.5 mM N -hexanoyltyramine with .025 mg tyrosinase. Best-fit line formul as are shown which form the ba sis for determining reaction and background rates. y = 4E-15x + 96.041y = -0.2739x + 119.49 y = -0.0065x + 96.869y = -0.2887x + 119.690 20 40 60 80 100 020406080100120140160180200%O2Time (s)1.75 mM N-Hexanoyltyramine .025 mg Tyrosinase y = -0.0477x + 109.84 y = -0.3547x + 140.84 y = -0.0094x + 101.76y = -0.2977x + 129.40 20 40 60 80 100 020406080100120140160180200%O2Time (s)2.5 mM N-Hexanoyltyramine .025 mg Tyrosinase

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57 Appendix D: Oxygen Electrode Traces for Lag Determination Figure D-1. Processed oxygen elect rode data for 6.6 mM tyramine with .025 mg tyrosinase. 0 20 40 60 80 100 020406080100120140160180200%O2Time (s)6.6 mM Tyramine .025 mg Tyrosinase

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58 Figure D-2. Processed oxygen electrode data fo r 6.6 mM tyramine with .05 mg tyrosinase. Figure D-3. Processed oxygen elect rode data for 6.6 mM tyramine with .075 mg tyrosinase. 0 20 40 60 80 100 020406080100120140160180200%O2Time (s)6.6 mM Tyramine .05 mg Tyrosinase 0 20 40 60 80 100 020406080100120140160180200%O2Time (s)5 mM Tyramine (EtOH) .075 mg Tyrosinase

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59 Figure D-4. Processed oxygen electrode data fo r 6.6 mM tyramine with .1 mg tyrosinase. Figure D-5. Processed oxygen electrode data fo r 6.6 mM tyramine with .15 mg tyrosinase. 0 20 40 60 80 100 020406080100120140160180200%O2Time (s)6.6 mM Tyramine .1 mg Tyrosinase 0 20 40 60 80 100 020406080100120140160180200%O2Time (s)6.6 mM Tyramine .15 mg Tyrosinase

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60 Figure D-6. Processed oxygen electrode data fo r 6.6 mM tyramine with .2 mg tyrosinase. Figure D-7. Processed oxygen electrode data for 1.2 mM N -acetyltyramine with .00625 mg tyrosinase. 0 20 40 60 80 100 020406080100120140160180200%O2Time (s)6.6mM Tyramine .20mg Tyrosinase 0 20 40 60 80 100 020406080100120140160180200%O2Time (s)1.2 mM N-Acetyltyramine .00625 mg Tyrosinase

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61 Figure D-8. Processed oxygen electrode data for 1.2 mM N -acetyltyramine with .0125 mg tyrosinase. Figure D-9. Processed oxygen electrode data for 1.2 mM N -acetyltyramine with .025 mg tyrosinase. 0 20 40 60 80 100 020406080100120140160180200%O2Time (s)1.2 mM N-Acetyltramine .0125 mg Tyrosinase 0 20 40 60 80 100 020406080100120140160180200%O2Time (s)1.2 mM N-Acetyltyramine .025 mg Tyrosinase

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62 Figure D-10. Processed oxygen electrode data for 1.2 mM N -acetyltyramine with .05 mg tyrosinase. Figure D-11. Processed oxygen electrode data for 1.2 mM N -acetyltyramine with .075 mg tyrosinase. 0 20 40 60 80 100 020406080100120140160180200%O2Time (s)1.2 mM N-Acetyltyramine .05 mg Tyrosinase 0 20 40 60 80 100 020406080100120140160180200%O2Time (s)1.2 mM N-Acetyryltyramine .075 mg Tyrosinase

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63 Figure D-12. Processed oxygen electrode data for 1.2 mM N -acetyltyramine with .1 mg tyrosinase. Figure D-13. Processed oxygen electrode data for 1.2 mM N -propanoyltyramine with .00625 mg tyrosinase. 0 20 40 60 80 100 020406080100120140160180200%O2Time (s)1.2 mM N-Acetyltyramine .1 mg Tyrosinase 0 20 40 60 80 100 020406080100120140160180200%O2Time (s)1.3 mM N-Propanoyltyramine .00625 mg Tyrosinase

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64 Figure D-14. Processed oxygen electrode data for 1.2 mM N -propanoyltyramine with .0125 mg tyrosinase. Figure D-15. Processed oxygen electrode data for 1.2 mM N -propanoyltyramine with .025 mg tyrosinase. 0 20 40 60 80 100 020406080100120140160180200%O2Time (s)1.3 mM N-Propanoyltyramine .0125 mg Tyrosinase 0 20 40 60 80 100 020406080100120140160180200%O2Time (s)1.3 mM N-Porpanoyltyramine .025 mg Tyrosinase

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65 Figure D-16. Processed oxygen electrode data for 1.2 mM N -propanoyltyramine with .05 mg tyrosinase. Figure D-17. Processed oxygen electrode data for 1.2 mM N -propanoyltyramine with .075 mg tyrosinase. 0 20 40 60 80 100 020406080100120140160180200%O2Time (s)1.3 mM N-Propanoyltyramine .05 mg Tyrosinase 0 20 40 60 80 100 020406080100120140160180200%O2Time (s)1.3 mM N-Propanoultyramine .075 mg Tyrosinase

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66 Figure D-18. Processed oxygen electrode data for 1.2 mM N -propanoyltyramine with .15 mg tyrosinase. Figure D-19. Processed oxygen el ectrode data for 2.3 mM N-butyryltyramine with .0031 mg tyrosinase. 0 20 40 60 80 100 020406080100120140160180200%O2Time (s)1.3 mM N-Propanoyltyramine .1 mg Tyrosinase 0 20 40 60 80 100 020406080100120140160180200%O2Time (s)2.3 mM N-Butyryltyramine .0031 mg Tyrosinase

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67 Figure D-20. Processed oxygen el ectrode data for 2.3 mM N-butyryltyramine with .0062 mg tyrosinase. Figure D-21. Processed oxygen el ectrode data for 2.3 mM N-butyryltyramine with .0125 mg tyrosinase. 0 20 40 60 80 100 020406080100120140160180200%O2Time (s)2.3 mM N-Butyryltyramine .0062 mg Tyrosinase 0 20 40 60 80 100 020406080100120140160180200%O2Time (s)2.3 mM N-Butyryltyramine .0125 mg Tyrosinase

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68 Figure D-22. Processed oxygen elec trode data for 2.3 mM N-butyryltyramine with .05 mg tyrosinase. Figure D-23. Processed oxygen el ectrode data for 2.3 mM N-bu tyryltyramine with .075 mg tyrosinase. 0 20 40 60 80 100 020406080100120140160180200%O2Time (s)2.3 mM N-Butyryltyramine .05 mg Tyrosinase 0 20 40 60 80 100 020406080100120140160180200%O2Time (s)2.3 mM N-Butyryltyramine .075 mg Tyrosinase

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69 Figure D-24. Processed oxygen elec trode data for 2.3 mM N-butyrylt yramine with .1 mg tyrosinase. 0 20 40 60 80 100 020406080100120140160180200%O2Time (s)2.3 mM N-Butyryltyramine .1 mg Tyrosinase


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Shafer, Jacob A.
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N-acyltyramines as substrates for tyrosinase
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b enzymatic lag and dopamine precursor /
by Jacob A. Shafer.
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[Tampa, Fla.] :
University of South Florida,
2009.
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Thesis (M.S.)--University of South Florida, 2009.
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Includes bibliographical references.
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Text (Electronic thesis) in PDF format.
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ABSTRACT: Tyrosinase is a widespread, highly studied and important enzyme involved in processes ranging from the browning of mushrooms to roles in mammalian cancer. The enzyme suffers from a noticeable lag phase while the enzyme generates all necessary cofactors from available substrates. There have not been significant studies of the effect on lag from moving through a family of substituted substrates. This thesis reports the results of one such study using a family of N-acyltyramines. The selection of N-acyltyramines was ideal because the substrates in this reaction may be related to synthesis of N-acyldopamines, which serve many important physiological functions. It was concluded that the product formed from N-acetyltyramine is 1-acetyl-2,3-dihydro-1H-indole-6,7-dione, a quinone.
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Mode of access: World Wide Web.
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Advisor: David J. Merkler, Ph.D.
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Kinetics
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Quinone
Mushroom
Oxygen electrode
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