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Tyrosinase-like Activity of Several Alzheime rs Disease Related and Model Peptides and their Inhibition by Natural Antioxidants by Kashmir Singh Juneja 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: Li-June Ming, Ph.D. Steven Grossman, Ph.D. Kirpal Bisht, Ph.D. Date of Approval November 13, 2006 Keywords: Alzheimers, Amyloid, Fl avonoids, Tyrosinase, Metzincins Copyright 2006, Kashmir Singh Juneja
Dedication This thesis is dedicated to the near 16 million people with Alzheimers disease. It is my hope that this work contributes to the understanding and ultimately treatment for this horrendous disease.
Acknowledgments My undergraduate mentor Vasiliki (Vaso) Lykourinou. I consider this woman a saint for maintaining sanity after being in the lab with so many children. Her patience and commitment is something that I am very envious and thankful for. William Tay, with the exception of the consis tent threats on my life, inability to make a lay-up, and horrible sense of direc tion, Tay has become a good friend who has provided nothing but positive support. Giordano da Silva, a boy amongst men, who trained under the finest Mongolian monk, has passed much of his knowledge on to me. Over the past five years he has constantly reminded me people dont hate me fo r the color of my ski n, its because of my personality. Erin Wu, for showing me the possibility to study abroad without ever learning the native language. I thank you for making me reali ze Labor Day falls on the day(s) I want it to. Dr. Ming, without meeting him my daily di et would still consist of subway, hot dogs, and microwave meals. With the ex ception of making me work with Gio, I appreciate Dr. Ming for everyt hing he has done for me, as it has helped me mature both mentally and culturally. My better half, Joumana Aram. For her devotion, tolerance, and support. And of course my parents, brother, a nd grandfather, for th eir support, without them, I would have never made it this far.
i Table of Contents List of Tables iii List of Figures iv List of Abbreviations vi Abstract vii Chapter One. Introduction 1 Enzymes 1 Metzincins 3 Copper Containing Enzymes 5 Amyloidand Alzheimers Disease 10 Flavonoids 12 Green Tea 15 Green Tea Catechins 15 Citrus Flavonols 18 Quercetin, fisetin, Taxifolin 18 Vitamins 20 Pyrdoxamine 20 Ascorbic Acid 21 Concluding Remarks 22 List of References 23 Chapter Two. Blastula Protease-10 Peptide as Tyrosinase-like Mimic 28 Introduction/Rationale 28 Experimental 29 Chemicals and Materials for Metal Titrations and Kinetics Assays 29 Peptide Preparation 29 Metal Binding 29 Enzyme Kinetics 31 Inhibition 41 Results and Discussion 42 Metal Binding 42 Catechol/Phenol Oxidation 45 Inhibition 53 Closing Remarks: 58 List of References 59
ii Chapter Three: Alzheimers Disease and Natura l Antioxidants 61 Introduction/Rationale 61 Experimental 63 Chemicals and Materials for Metal Titrations/Kinetics Assays 63 Peptide Preparation 63 Dopamine and Flavonoid Oxidation assays 63 Inhibition Experiments 64 Results Discussion 66 Green Tea 66 Quercetin, Fisetin, and Taxifolin 77 Closing Remarks 85 List of Referneces 86
iii List of Tables Table 1-1. Information on metalloenzymes 2 Table 1-2: The classifica tion and structure of severa l well studied flavonoids 14 Table 2.1: Kinetic parameters for H2O2 effect on Cu2+-BP10 50 Table 3-1: Molar Absorptivity values for neurotransmitter and flavonoids 64 Table 3-2: Apparent and intrin sic affinity constants for A 16 and A 20 72
iv List of Figures Figure 1-1: Diagram of the zinc environment in the metzincins.2 4 Figure 1-2: The role of tyrosi nase in the production of melanin.8 7 Figure 1-3: Proposed intermediate s for phenol hydroxylation 8 Figure 1-4: Proposed m echanism for tyrosinase.9 9 Figure 1-5: Amino aci d sequence of Amyloidpeptides (A ). 11 Figure 1-6: The green tea catechins 16 Figure 1-7: Structure of flavonols 19 Figure 1-8: Structures of pyridoxamine and pyri doxamine-5-phosphate 21 Figure 1-9: Structure of ascorbic acid 22 Figure 2-1: Scheme showing the binding of o-qunione indicator MBTH 30 Figure 2-2: Michaelis-Menten plot 33 Figure 2-3: Lineweaver-B urk plot. 34 Figure 2.4: Graphical, schematic, and e quations for competitive inhibition 35 Figure 2.5: Noncompetitive inhibition 36 Figure 2.6: Mixed-type inhibition. 37 Figure 2.7: Uncompetitive inhibition 38 Figure 2-8 (A) Absorption of the MBTH adduct 40 Figure 2-8 (B) Increase in absorpti on for catechol oxidation. 40 Figure 2.9: Electronic spectra of Cu2+-BP10 43 Figure 2.10: Metal titration and Zn-dilution of BP10 44 Figure 2.11: Cu2+-BP10 oxidation of catechol 46 Figure 2.12: The effect of H2O2 on Cu2+-BP10 oxidation of catechol 47 Figure 2.13: Random bisubsubstrate e quation and equilibrium 48
v Figure 2.14: Effect of [H2O2]on kcat toward the Cu2+-BP10 oxidation of catechol. 49 Figure 2.15: Hanes analysis of Cu2+-BP10 oxidation of catechol with H2O2 51 Figure 2-16: Cu2+-BP10 hydroxylation/oxidation of phenol and d-phenol 52 Figure 2-17: Kojic Acid Inhibition 54 Figure 2-18: Cyanide Inhibi tion in the presence of O2. 55 Figure 2-19: Cyanide Inhibi tion in the presence of H2O2, varying H2O2. 56 Figure 2-20: Cyanide Inhibi tion in the presence of H2O2, varying catechol. 57 Figure 3-1: Purposed mechanis m for polyphenol oxidation by Cu2+-A 62 Figure 3-2: A 16 oxidation of dopamine, EC, EGCG 67 Figure 3-3: A 20 oxidation of dopamine, EC, EGCG 68 Figure 3-4: A 16,20 oxidation of epigallocatechin. 69 Figure 3-5: Effect of [H2O2]on kcat toward the Cu2+A 16 oxidation of substrates 71 Figure 3-6: Effect of [H2O2]on kcat toward the Cu2+A 20 oxidation of substrates 71 Figure 3-7: Inhibition of Cu2+-A 1-16 by ascorbic acid 73 Figure 3-8: Inhibition of Cu2+-A 1-20 by ascorbic acid 74 Figure 3-9: Inhibition of Cu2+-A 1-20 by Pyridoxamine 75 Figure 3-10: Inhibition of Cu2+-A 1-16 by Pyridoxamine 76 Figure 3-11: Quercetin inhibition of Cu2+-A 1-16 79 Figure 3-12: Fisetin inhibition of Cu2+-A 1-20 80 Figure3-13: Fisetin inhibition of Cu2+-A 1-16 81 Figure 3-14: Ca2+ effect on Fistein inhibition of Cu2+-A 1-16 82 Figure3-15: Taxifolin inhibition of Cu2+-A 1-16 83 Figure3-16: Taxifolin inhibition of Cu2+-A 1-20 84
vi List of Abbreviations A 16 Beta-amyloid 16 amino acid A 20 Beta-amyloid 20 amino acid AsA Ascorbic acid BP10 Blastula Protease 10 EC Epicatechin EDTA Ethylenediamine tetraacetic acid EGC Epigallocatechin EGCG Epigallocatechin gallate GTC Green Tea Catechin HEPES N-[2-Hydroxyethyl]piperazi ne-N-[2-ethanesulfonic acid] MBTH 3-methyl-2-benzothiazo linone hydrazone hydrochloride monohydrate ROS Reactive oxygen species
vii Tyrosinase-like Activity of Several Alzhei mers Disease Related and Model Peptides and their Inhibition by Natural Antioxidants Kashmir Singh Juneja ABSTRACT Neurodegenerative diseases are associated with loss of neurons ultimately leading to a decline in brain function. Alzheimers disease (AD) is considered one of the most common neurodegenerative disorders that a ffects 16 million people worldwide. The cause of the disease remains unknown, alth ough significant evidence proposes the amyloid -peptide (A ) as a potential culpri t. The binding of Cu2+ by the soluble fragments of A have shown to form Type-3 copper centers and catalyze the oxidation of catechol-containing neurotransmitters. Furtherm ore, the use of flavonoids as antioxidants to slow or inhibit the neurotransmitter oxid ation has suggested further health benefits with their consumption. A structure-func tion correlation is also made between the flavonoids and their reactively with Cu2+-A Mechanistic insight into the binding of catechol and dioxygen within the tyrosinase-like mechanism are made using a metallopeptide modeling the active site of the metzinicins.
1 Chapter One Introduction Enzymes Enzymes are essential proteins that ha ve the ability to regulate and govern numerous reactions required fo r life. They serve as biolog ical catalysts, reducing the energy barrier in a reaction. The catalytic proficiency is furt her enhanced by an enzymes ability to be substrate specific. In general, enzymes can be categorized on the basis of the type of reaction in which they perform. Examples include oxidoreducatses, hydrolases, and transferases. The catalysts that fall under the oxidor educatase category are involved in redox reactions. These redox reactions involve the tr ansfer of electron(s) from one species to another.1 Redox reactions are involved extensively in industrial application, humus degradation, and are essential for life on this planet. Biological systems use these oxidoreductases in anabolism, catabolis m, protective, and energy sublimentive functions.2 Being that the inside of the cell is under reductive conditions, these enzymes are used to regulate and specify when and where a redox reaction takes place. In biological systems, constitutes formed are sometimes the result of several enzymes. Whether the product is modified or the enzyme is regulat ed, it is usually a cascade of reactions that is i nvolved in synthesizing the necessa ry biological components. Hydrolases are another class of enzymes that activate a water molecule to serve as a nucleophile in a substrat e-specific bond cleavage.2 These hydrolytic enzymes are further classified on the basis of their substrate spec ificity. An example is endopeptidases which
2 cleave peptide bonds within a peptide or protei n at specific locations other then C and Nterminal domains. The structure of the protein, specifically the active site, controls the specificity of the enzyme. In many enzymes, metals ions can be found within the active site to assist in catalysis. Transition metals are excellent Lewis acids that have the ability to carry a charge and still contain a high electron affinity.3 In an effort to continue catalysis, metal ion(s) undergo a degree of mobility by making sli ght changes in its coordination during a reaction. The differences in me tal ions allow each to prefer particular geometries and types of chemistry.3 Table (1-1) summarizes inform ation about several well-known metalloenzymes. Table 1-1. Information on some metalloenzymes. Metalloenzyme Metal Ion(s) Occurrence Function Reverse Transcriptase3 Zn Human immunodeficiency virus (HIV) Transcribes ssRNA into dsDNA Tyrosinase3 2 Cu Plants and Animals Hydroxylation and oxidation of phenol Lipoxygenase3 Fe Animals Catalyse the dioxygenation of polyunsaturated fatty acids Methionine aminopeptidase3 2 Co Bacteria to Animals Removal of N-terminal methionine Urease3 2 Ni Jack Bean and bacteria Hydrolysis of urea to ammonia Mn-catalase3 2 Mn Prokaryote Decomposition of 2 H2O2 2 H2O + O2 Bromoperoxidase3 V Some brown & red marine algae Defensive Mechanism Chromodulin4 Cr Human Unknown, possible insulin signaling DMSO reductase3 Mo Bacteria Dimethyl sulfoxide to dimethyl sulfide Acetylene hydratase3 W Pelobacter acetylenicus Hydration of acetylene to acetaldehyde
3 Metzincins The function of a metalloenzyme can be related to the transition metal ion(s) within its active site. Of the transition metals zinc is one of the most readily available to biological systems, ranging from 10-11 to 10-3 M in various portions of a cell.3 Zn(II) ion has the electronic conf iguration of [Ne] 3d10, lacking both spectroscopic and magnetic properties. Like many of the first row transi tion metals, zinc is often found in divalent state (Zn2+) because of the loss of the 4s2 electrons. When consid ering divalent cations, Zn2+ is an excellent Lewis acid, second only to Cu2+.3 The unique properties of Zn2+ also include extremely flexible coordination geom etry extending, from 4 to 6 coordination. The most common ligands for Zn2+ are thiolate, imidazole, wa ter, and carboxylate. The Zn2+ found in metalloenzymes can serv e a structural role or be involved in the reaction. For example, the Zn2+ in Cu,Zn-superoxide dismutase se rves a structural role that stabilizes the protein.3 In other cases Zn2+ is involved in reactions, where in the metalloenzymes most always perform hydro lysis. The classification of these hydrolytically active Zn2+ enzymes is based on the ligands coordinated to the metal ion and the substrate specificity. For the past two decades, several large groups of Zn2+-containing enzymes have received much attention because of similarities in their structure and distinctive location. The following groups have been classified as zinc endopeptidases: astacins, adamalysins, serralysins, matrixins.5 These endopeptidases contain a common -helical Zn2+ binding motif (HEXXHxxGxxH) and a distant methionine turn (Figure 1.1). It is because of these similarities that all of these families have been grouped into one super family called the metzincins.5 Despite their common structure, th e metzincins have been found in
4 numerous locations including caryfish dige sitive fluid, sea urchin embryos, and snake vemon.5,6 In the metzincins, the metal is coor dinated by 3 His side chains and a water molecule which is H-bonded to the Glu in the motif.5 Most recent evidence reveals a distant Tyr after the Met-turn in astacin, wh ich stabilizes the enzyme-substrate complex through H-bonding and relieves steric hindrance.7 In addition, several studies have shown accelerated hydrolytic activit y upon substitution of the native Zn2+ with Cu2+ or Co2+.7,8 It is evident through the properties and abundance of Zn2+ that this unique transition metal is one of the most important in biological systems. The flexibility and ligand exchange rate ha veforced nature to develop a dyna mic scheme of delivery of this precious metal.3 Metal substitution experiments have postulated natures use of Zn2+ instead of another transition metal beca use of its inertnes s in redox chemistry. Figure 1-1: Diagram of the zinc environment in the metzincins.5
5 Copper-Containing Enzymes Copper-associated chemistry is very rich in nature. Exceeding all other transition metals, Cu2+ is a very effective divalent i on for binding organic ligand molecules.3 The high electron affinity makes it a valuable a sset in biological redox chemistry. Several Cucontaining enzymes can bind and activate small molecules such as O2.3 It is the affinity for these molecules and large redox potential that has forced nature to developed specialized transport systems to maintain homeostasis and limit free Cu2+ to 10-18 M in the human body.9 To replenish the body, it is recommended to consume 0.9 mg of copper per day.9 Copper is absorbed mainly in the small inte stine and transported to the liver. Here, transporters and chaperons deliv er the metal to various locati ons in the body. One of the main transports is human copper transport protein (hCtr1). 9 Together with the influx of potassium (K+), copper is taken up and delivere d to several chaperons or storage structures such as th e metallothionein pool.3 The chaperons in turn supply Cu to proteins like superoxide dismutatse, amyloi d precursor protein (APP) dopamine -hydroxylase, and tyrosinase. The role of many Cu enzymes is O2 activation followed by oxidation of a substrate.10 The mechanistic differences within Cu-proteins are due to the protein structure, the number of Cu ions and the coordination chemistry. The copper within proteins is usually limite d to one of three types of coordination. Each copper protein can be categorized as T ypes I-III. Type I copper proteins are wellknown for their intense blue color and consist of blue Cu-proteins and blue Cuoxidases.11 The blue Cu-proteins contain one c opper ion coordinated by two histidines, one cystein, and one loosely coordinated methionine in a trigonal or trigonal bipyrimadal
6 conformation.11 An example of a Type I copper prot ein is the electron transfer protein plastocyanin in photosynthesis. The active site of Type II copper protein is usually coordinated by both nitrogen a nd oxygen-containing ligands in a tetragonally distorted configuration.11 A well-known Type II copper enzyme is the radical scavenging Cu/Znsuperoxide dismutase. The third group of c opper proteins are the EPR silent Type III copper proteins. These copper proteins cont ain two copper ions as a dinuclear center coordinated by six histidine residues.11 One of the best known examples is tyrosinase. To date, tyrosinase is considered one of the most well studied multicopper oxygenases. Found widely in living systems, ty rosinase is responsible for the preliminary steps in the synthesis of melanin.12 Like all Type III copper pr oteins, tyrosinase utilizes its dinuclear center to bind dioxygen. Following the subsequent activation of O2, it hydroxylates and oxidizes the phenolic substr ate to yield the ortho-quinone product (Figure 1-2).12 The rates for the oxidation (107 s-1) is ten thousand times that of the hydroxylation (103 s-1).11 To determine the mechanism and its intermediates, nitrogenbased model systems have been used extensively.13
7 The reaction at the dinuclear center of tyrosinase begins with the binding of dioxygen, converting the deoxy into the oxy form of the dinuclear center. Monophenol then binds to one of the copper centers, al lowing for it to be oriented for ortho hydroxylation. The hydroxylation is believed to go through one of three intermediates O H O H NH3+ COOTyrosinase O2H 2O O O NH3+ COONH2+ O O COONH2+ O H O H COONH2+ O O L-dopa Dopaquinone Leucodopachrome Dopachrome Eumelanin NH3+ COOO H Tyrosinase 5,6-dihydroxyindole Figure 1-2: Scheme depicting tyrosinase activity in the production of melanin.12
8 (Figure 1-3).11 One intermediate involved the oxyge n bridge cleavage prior to attack, resulting in the forma tion of a binuclear Cu3+.11 The second is the breakage of the oxygen bridge with the attack.11 And lastly, is a possible aryl peroxide intermediate.11 The resulting diphenol is bound to the met-D center (Figur e 1-4), allowing for a twoelectron oxidation to form the o-quinone. In addition to the monophenolase activity, tyrosinase can oxidize catechol (diphenols) di rectly. Both the met and oxy forms of the dinuclear center can bind and promote the ox idation of catechol. The reaction continues in this cycle until the substrate has been depleted or the enzyme is inhibited. O N N O O Cu3+N N N N Cu3+ N N Cu2+O O Cu2+N N N N O O O O Cu2+N N N N Cu2+ N N Figure 1-3: Three proposed intermediate s for the hydroxylation of phenol by tyrosinase.11
9 The intermediates and mechanism for ty rosinase were solved using various synthetic metal complexes as model systems. The structure of these complexes varies but generally contain N-based f unctional groups such as amin e, pyridyl, pyrazolyl, and imidazole.14 Through the use of numerous spectrosc opic techniques and low-temperature experiments, a number of plausible Cu:O2 intermediates have been found.10 However, N N Cu2+O O Cu2+N N N N Cu2+O O Cu2+N N O O O N N Cu2+ OH Cu2+N N OH H+ N N Cu+ H+ O O H2 O O2 O O N N Cu2+ OH Cu2+N N O OH OH O O H2 O H N N Cu2+ OH Cu2+N N OH OH Cu+N N Oxy-T Oxy met-D deoxy + oxy-D H 2 + + + 3 met H 2 + Monophenolase Cycle Diphenolase Cycle Figure 1-4: Proposed m echanism for tyrosinase.11
10 these model systems have been shown to contain reduced tyrosi nase activity. The modeling of active sites for activ ity and binding is an ever gr owing trend that extends to far beyond just Type III copper proteins.15 Amyloidand Alzheimers Disease Through advances in modern medicine, th e duration of life has been extended by eliminating or postponing vari ous human diseases. Unfortunately, with the average life span almost doubling from the 19th century there has been a significant increase in agingrelated illnesses.16 Neurological disorders such as Alzheimers, Parkinsons, and Huntingtons disease, have caused increase d concern for the ever-growing number of victims. The most common neurodegenerative disease is Alzheimers (AD), affecting near 4.5 million Americans.17 With only 10% of the cases being familial AD, the majority of the occurrences are s poradic and currently unpredictable.17 In general, a neurodegenerative disease is associated with the accumulati on of misfolded or fragmented protein that affects normal neuronal function.9 AD is a progressive neurodegenerative di sease that causes memory and motor skill loss. There are three hallmarks associ ated with AD which are believed to be responsible for the loss of neuronal function, located prim arily in the hippocampus and cortex: (a) accumulation of ne urofibrillary tangles compos ed of the hyperphosporylated microtububle-associated tau protein (p-tau), (b) insoluble plaques formed from the amyloidpeptides (A ), and (c) ramped loss of neurons.9,18 Even though the exact cause of AD is still unknown, many have hypothesized an amyl oid cascade leading to all three of the hallmarks.
11 Even with slight variatio ns, it has been agreed upon that the abnormal processing of the transmembrane amyloid precursor protein (APP) causes an increase in the production of A .11,17 This overproduction is believed to affect synapses, causing altered ionic and enzymatic homeostasis resulting in tangles, plaques, and ultimately cell death.9 The order and location of cl eavage by three secreatases ( , ) determine whether the product will be considered am yloidogenic or nonamyloidogenic.9 The nonamyloidogenic pathway begins with the -secreatase cleavage followed by a -secreatase forming a shorter more soluble fragement of A .9 The amyloidogenic pathway is initiated by secreatase followed again by -secreatase.9 The fragments of APP following cleavage range from 16-42 amino acids in length (Figure 1-5), with the insoluble A 40 and A 42 believed to have the largest effect on neuronal cell loss.9,18 In addition to the accumulation of protei n fragments, postmortem studies have reported millimolar amounts of Zn2+, Cu2+, and Fe3+ within the amlyoid plaques.17 The findings of redox-active metals have fuel ed the hypothesis of reactive oxygen species (ROS) as a major contributor to the degrad ation of brain functi on in AD. The ROS species normally generated by the body are us ed for degradation and defense purposes.2 The body regulates ROS by both SOD and catalase The hypothesis that ROS is part of AD is well justified as non-regulated accumula tion of redox active metal has led to other DAEFR5HDSGY10EVHHQ15KLVFF20AEDVG25SNKGA30IIGLM35VGGVV40 IA42 Figure 1-5: Amino aci d sequence of Amyloidpeptides (A ).
12 illnesses such as Wilsons Disease (WD).9 The metal-centered ge neration of ROS is believed to be consistent with the Fe nton and Haber-Weiss reactions shown below.9 Studies have shown that APP is an active participant in copper homeostasis, with significant loss of this protein show ing elevated levels of free Cu2+.9 Not surprising is that A has also shown to chelate metal with a high affinity.19 Through the use of NMR, the binding site for the metal has shown to be three His within the first 14 amino acids.20 Additional studies have shown the possi ble dimerization and coagulation of A to begin at amino acid 17-20.21 Although much emphasis has been put on A 40,42 numerous structure studies are focused on all of the A and possible ways to inhibit its formation. To date, treatments for AD include meta l chelators and acetylcholine esterase inhibitors. Unfortunately ther e are many side effects associated with the metal chelators, specifically due to the chelation of needed metal ions. The binding of redox-active metals to solvent-exposed peptide domains has raised the issue of possible ROS generation in AD. This emphasizes the develo pment of bioavailabile metal chelators or the use of antioxidants to scavenge ROS. Flavonoids In ancient China, there had been evidence of the use of antioxidants as a remedy to cure human illness. Of these antioxida nts, a group of phenolic plant constituents encompass a major portion of those cons umed around the world for their potential benefits. To date, ther e are over 6000 of these compounds known as flavonoids.22 O2 + H2O2 OHOH + O2 (Haber-Weiss reaction) Mn+ + H2O2 OH+ OH + M(n+1)+ (Fenton reaction)
13 Several clinical studies have been done concerning the possible protection against cancer, cardiovascular, and neurodegenerative diseases.23,24 They have been further used for their potential anti-fungal, anti-microbi al, and anti-radical properties.22 Flavonoids have gained much attention over the years because of their potent antioxidant properties and bioa vailability. The structural di fferences of the flavonoids, although subtle, have shown to rema rkable change their bioactivity.25 It is these differences that allow the flavonoids to be divided into subcategories. The general structure consists of two benzene rings (A and B) linked though a tetrahydropyran or pyrone ring (C).22 Flavones (e.g. apigenin) contain a double bond at the 2-3 position, while flavanones (e.g. narigenin) are staturat ed at this position. A double bond at the 2-3 position and a hydroxyl, methoxy, or sugar at the 3 position represents the flavonol category (e.g. quercetin, fise tin). Dihydroflavonols contai n a hydroxyl group at the 3 position and is absent of the 2-3 double bond. The catechins lack the ketone functionality in the C ring and contains hydroxyl groups at 3, 3, and 4 positions. Many other classifications exist for flavonoids that contain further unsatutartion, hydroxylation, epoxidation, and sugar modification. Table 12 describes the structure of several well studied flavonoids. The quantity of each group of flavonoids depends on the kind of plant, climate, and location the plant is found. For exampl e, several categorie s are found in higher amount in citrus, while others ar e found in green-leaf vegetables.22 Although there is an abundance of flavonoids within the diet, their protective properties are only good as they are absorbed. Several flavonoids have better absorptive properties then others. It has
14 Flavonoids Classification R1 R2 R3 R4 R5 R6 2-3 Alkene 4 Ketone (-)-Epicatechin Flavan-3-ol H OH OH OH OH H (-)-Epigallocatechin Gallate Flavan-3-ol Gallate OH OH OH OH OH Fisetin Flavonol OH H OH OH OH H + + Quercetin Flavonol OH OH OH OH OH H + + Taxifolin Dihydroflavonol OH OH OH OH OH H + Apigenin Flavone H OH OH H OH H + + Narigenin Flavone H OH OH H OH H + Hesperetin Flavanone H OH OH OH OCH3 H + Rutin Flavonol glycoside Rutinose OH OH OH OH H + + been shown that lactase and -glycosidase can cleave the gl ucoside portion off the sugar derivatives of flavonoids.22 It is the effects following ab sorption that has increased the interest in the natural polyphenols. With numerous illnesses and disease being a ssociated with ROS, the antioxidant and antiradical properties of flavonoids have b ecome the center of attention. For a compound to be considered a strong antioxidant it mu st inhibit oxidation reactions and/or the production of radicals at a lo w concentration compared to the oxidizable substrate. Table 1-2: The classifica tion and structure of severa l well studied flavonoids. A B C3'5'4' R 3 O R 1 O R 5 R 4 R 6 R 2 7 5 4 3 2
15 Furthermore, the radicals formed by flavonoids must be stable enough not to continue in as a chain propagating radical. These properties associated with flavonoids have been used in conjunction with other molecules to further stabilize or complement the flavonoids bioactivity.26 Green Tea Believed to have originated some 3000 year s ago in ancient Chin a, tea is now one of the most consumed beverages in the world.27 The leaf extract of the plant Camellia sinensis, also known as tea, have shown to be rich in antioxidant polyphenols, ascorbic acid, and trace elements Cr, Mn, Se, and Zn.27 Depending on the species, season, and extent of fermentation, the amounts of these health-beneficial compounds can vary significantly. The trace elements Mn, Se, Zn are directly involved with a number of enzymes that reduce oxidative damage.3 Biological systems use Mn as a constituent for Mn-superoxide dismutase. Additionally, Se serves as a cofactor for glutathione peroxidase, allowing for the re moval of peroxide radicals.3 When considering green, oolong, and fermented teas, green tea has shown to contain a higher content of catechins and other hydroxylated phenols.27,28 Within green tea, the ge neral trend of quantity of green tea catechins (GTC) is (-)-epigallocatech in gallate (EGCG) > (-)-epicatechin gallate (ECG) > (-)-epicatechin (EC) (-)-epigallocatechin (EGC) >> (+)catechins.27,28 Green Tea Catechins (GTC) The GTCs are similar in structure, differing only by as many as 2 substitutions (Figure 1-6). The structure of ECG and EC differ only by a gallate pr esent on position 3. EGCG and EGC differ only by an additional hy droxyl group on the B ring in position 5. Despite these small differences, studies have shown them to differ in various types of
16 bioactivity and availability. Like many flavonoi ds, the GTCs have been shown to exhibit potential protective effects against cardiovasc ular disease, cancer and neurodegenerative disease.28 O O O OH OH OH OH OH OH OH O H O OH OH OH OH OH O H O OH OH OH OH O H (A) (B) (C) Figure 1-6: The green tea catechins (A) Epicatechin (EC), (B) Epigallocatechin (EGC), (C) Epigallocat echin Gallate (EGCG).
17 GTCs have gained popularity as they ha ve been demonstrated to show metal chelating, free radical scavenging, protein inter action, and transcripti on factor regulatory abilities.23 Specifically, several links have b een made between GTCs and diseases involving ROS. Following their reactions wi th free radicals GTCs form a number of dimers and seven member anhydride rings.29 In comparison w ith the bodys natural radical scavengers, EGCG can increase cell survival similar to that of catalase in ROS affected cells.30 Structurally, implications have been made on the advantage of the trihydroxybenzene and gallate moieties to enhance the anti oxidant and metal chelation abilities.23,28 In addition to its chemical properties, the brain-permeability of GTCs may offer beneficial effects in several neur odegenerative diseases. A recent study on Alzheimers disease has linke d EGCG with APP processing.31 It was shown in vivo and in vitro that EGCG enhances the activation of the -secretase and inhibits -secretase activity, leading more toward a nonamyloidogenic pathway.31 Despite their reactivity, there is much concern over the stability of GTCs. Following an oral dose of 100mg of GTCs, only 9-10ug/ml will be absorbed.26 The absorption deficiency may be due to the cha nge from the acidic stomach to the alkaline blood.26 In basic conditions, the trihydroxyben zene is probably more susceptible to oxidation and the gallate is hydrolytic ally cleaved to form gallic acid.26 Despite these absorption problems, green tea remains one of many good sources of flavonoids.
18 Citrus Flavonols Citrus is a flowering plant genus found in the Rutaceae fam ily. It includes fruits such as oranges, grapefruits, and lemons. There is an annual pr oduction of 80 million tons of citrus fruits world wide.32 These fruits are known for th eir characteristic scent and sharp taste. They are rich both in vitami ns and flavonoids. Citrus has utilized these compounds to develop pigmentation and prot ection from insects in addition to ROS.22 Studies have linked the compone nts in citrus to prevention of cardiov ascular disease, cancers, and allergies.33 Depending on the fruit, citrus can be an excellent source of many flavonoids. Three structurally simila r polyphenols found in citrus are quercetin, fisetin, and taxifolin, which vary in medicinal effect. Quercetin, Fistein, and Taxifolin In general, there are several presumed structural requirements for flavonoids to have good antioxidant/antiradical properties. The structural requirements include: a catechol/polyphenol B ring, 2-3 double bond, abundance of free hydroxyl groups, and specifically the 3-hydroxyl group.34 Quercetin is the most common flavonol in the human diet. There is an abundance of quercetin in onions, fr uits, teas, and red wine.22 Variations of quercertin are found naturally, having one or more sugars bound at the 3 position. Studies have shown these sugar moieties assist in the absorption of quercetin.22 Like many flavonoids, queretin bind metal in addition to scavenging free radials.22,35 Fisetin is a le ss common flavonol, found in various fruits and vege tables. It differs from que rcetin in that it lacks a phenolate. The desaturation of quercetin at th e 2-3 position yields ta xifolin, appropriately known as dihydroquerctein. Taxifolin is considered a dihydroflavonol and is also found in some fruits and vegetables.34
19 Many studies have compared the flavonoids based on their level of antioxidant and antiradical activity. In a st udy published by Oleszek et al.(34) the antioxidant properties were found to increase with the presence of the 2-3 double bond (i.e., Quercetin > Fisetin >> Taxifolin). They also showed the antiradi cal properties were affected by the presence of the 3-OH and not the 2-3 double bond (Taxifolin > Quercetin > Fisetin).34 As there has been some debate over the best fl avonoid, it generally agreed upon that the combination of several antioxidants would yiel d the best antioxidant /radical properties. O OH OH O OH O H OH O OH OH O OH O H O OH OH O OH O H OH (A) (B) (C) Figure 1-7: Structure of flavonols qercetin (A), fise tin (B), and dihydroflavonol
20 Vitamins Once thought to require an amine functional group, they were termed vital amines (vitamine). Over the years, structural evidence revealed the lack of the amine in many vitamines, resulting in the loss of the e (vitamin).36 Most vitamins are obtained through the diet and are classified on either being water (B an d C) or fat (A, D, E and K) soluble. These compounds are found in abu ndance in human diet, with fruits and vegetables being excellent s ources. Studies have shown that many vitamins have excellent antioxidant proper ties and are directly involved in many human illnesses.36 Vitamin B6 Considered to be 1 of 8 components of the vitamin B complex, vitamin B6 is found in three structurally distinct form s: pyridoxal, pyridoxine, pyridoxamine. An enzyme known as pyridoxal kinase converts e ach of the three in the active form of vitamin B6, pyridoxal 5-phosphate.37 The body utilizes the active form as a cofactor for over 140 enzymes.37 Some of which are involved in amino acid and monoamine neurotransmitter synthesis.38 A deficiency in vitamin B6 has been shown to lead to insufficient insulin and altered hormone production.38 The recommended daily intake is 2 mg, which is easily obtained from various vegetables, fish, and non-citrus based fruit.38 In addition to its regulatory roles vitamin B6 has also been shown to serve as a potent antioxidants.39 Studies have suggested th at components of vitamin B6 inhibit the product of radicals and serve as que nchers for singlet oxygen.39
21 Ascorbic Acid Being the most abundant sol uble antioxidant in plants L-ascorbic acid (AsA) (aka. Vitamin C) (Figure 1-9) has become increasingly consumed because of its proposed health benefits.40 At risk of diseases such as scur vy, AsA is considered essential in the human diet. The body uses AsA as radical scavenger, calcium regulator, and as a cofactor for multiple enzymes, some involve d in collagen synthesis. It is the antioxidant/antiradical properties associated w ith AsA have believed to be responsible for its contribution to the prevention of numerous chronic diseases.40 Through consumption of beverages such as green tea, one can easily obtain the recommended daily intake of 30-110 mg/day.40 In addition to its health benefits, AsA effect on the absorption of other biologi cal components has also been measured.26 For example, the low absorption of GTCs is thought to be because of its oxidative breakdown. Results have shown AsA to serve as a reductant that can protect GTCs and potentially increase their total absorption.26 Although the overall e ffect of one compound N CH3 OH N H2 O H N CH3 OH N H2 Phosphate Figure 1-8: Structures of pyridoxamine and pyridoxamine-5-phosphate
22 can be significant, it is usually thought that a combination of antioxidants (e.g. flavonoids and vitamins) can provide a more bene ficial antioxidant protective effect.41 Closing Remarks: When characterizing an enzyme, it is not uncommon to use model systems to reveal both mechanistic and structural informa tion. Furthermore, it is often beneficial to find stable enzyme mimics that exhibit high le vels of activity. This thesis presents both modeled and natural peptides that show tyrosinase-like activity. The former is the metzinicin active site and is characterized throug h metal binding, activity, and inhibition. The latter are varied fragments of Alzheimers disease-related amyloid. Catalytic efficiency and mechanistic insight are obtained on amyloi d through the use of physiologically relevant substrates. In additi on, select flavonoids and vitamins are used to show that the possible consumption of high content antioxida nt foods can reduce oxidative stress caused by A These antioxidants are compared based on their overall effect of the A tyrosinase-like chemistry. O O OH O H O H O H Figure 1-9: Structure of ( R )-3,4-dihydroxy-5-(( S )-1,2-dihydroxyethyl)furan-2(5 H )-one (Ascorbic Acid)
23 References: 1) McMurry, J.; Fay, R. Chemistry 3rd Edition; Prentice-Hall: New Jersey, 2001. 2) Voet, D.; Voet, J. Biochemistry, 3rd Edition; John Wiley & Sons: New Jersey, 2004. 3) da Silva, F., and Williams, R. The biological chemistry of the elements. 2nd edition. Oxford University Press, 2000. 4) Vincent, B. (2000) Elucidat ing a Biological Role for Chromi um at a Molecular Level. J. Acc. Chem. Res., 33, 503-510 5) Wolfram, B., Franz-Xaver, G., and Walte r, S. (1993) Astacins, serralysins, snake venom and matrix metalloproteinases exhi bit identical zinc-binding environments (HEXXHXXGXXH and Met-turn) a nd topologies and should be grouped into a common family, themetzincins FEBS Letters., 331, 134-140. 6) da Silva, F. Z. G., Reuille, L, R ., Ming, L., and Livingston, T, B. (2006) Overexpression and Mechanistic Charact erization of Blastula Protease 10, a Metalloprotease Involved in Sea Urchin Embryogenesis and Development. J. Biol. Chem., 281, 16, 10737-10744. 7) Hyun, P. and Ming, L. (1998) The mechanistic role of the coordinated tyrosine in astacin. J. Inorganic Biochem., 72, 57-62. 8) Hyun, P. and Ming, L. (2002) Mechanistic studies of the astacin-like Serratia metalloendopeptidase serralysin: highly act ive (>2000%) Co(II) and Cu(II) derivatives for further corroboration of a "metallotriad" mechanism. J. Biol. Inorg. Chem., 7, 600610.
24 9) Gaggelli, E,. Kozlowski, H,. Valensin D,. and Valensin G. (2006) Copper homeostasis and neurodegenerative disord ers (Alzheimer's, prion, and Parkinson's diseases and amyotrophic latera l sclerosis). Chem. Rev., 106, 1994-2044. 10) Mirica, M, L., Ottenwaelder, X., and Stack, P, D. (2004) Structure and spectroscopy of copper-dioxygen complexes Chem. Rev., 104, 1013-1045. 11) Soloman, I, E., Sundaram, M, U., and Machonkin, E, T. (1996) Multicopper Oxidases and Oxygenases Chem. Rev. 96, 2563-2605. 12) Ferrari,P., Laurenti, E., Ghibaudi, M, E ., and Casella, Luigi. (1997) Reversible Dioxygen Binding and Phenol O xygenation in a Tyrosinase Model System. J. Inorg. Biochem., 68, 61-69. 13) Selmeczi, K., Reglier, M., Giorgi, M., and Speier,G. (2003) Catec hol oxidase activity of dicopper complexes with N-donor lig ands. Coordination Chem. Rev., 245, 191-201. 14) Mahadevan, V., Gebbink, K, R., and Stac k, T, D. (2000) Biomimetic modeling of copper oxidase reactivity. Curr. Opin. Chem. Bio., 4, 228-234. 15) Boka, B., Myari, A., Sovago, I., and Ha djiliadis, N. (2004) Copper(II) and zinc(II) complexes of the peptides Ac-HisValHis-NH2 and Ac-HisValGlyAsp-NH2 related to the active site of the enzyme CuZnSOD. J Inorg Biochem 98,113-22. 16) CDC.gov 17) Huang, X., Moir, D, R., Tanzi, E, R., Bush, I, A., and Rogers, T, J. (2004) RedoxActive Metals, Oxidative Stre ss, and Alzheimer's Disease Pathology. Ann. N.Y. Acad. Sci. 1012, 153-163. 18) Chong, Z, Z., Li, F., and Maiese, K. (2005) Employing New Cellular Therapeutic Targets for Alzheimer's Disease: A Change for the Better?. Curr. Neurovascular Research, 2, 55-72.
25 19) da Silva, F, Z, G., Tay, M, W., and Ming, L. (2005) Catechol Ox idase-like Oxidation Chemistry of the 120 and 116 Fragment s of Alzheimer's Disease-related -Amyloid Peptide. J. Bio. Chem., 280, 17, 16601-16609. 20) Mekmouche, Y., Coppel, Y., Hochgrfe, K., Guilloreau, L., Talmard, C., Mazarguil, H., and Faller, P. (2005) Characterization of the Zn II Binding to the Peptide Amyloid-b linked to Alzheimers Disease. Chem. Bio. Chem., 6, 1663-1671. 21) Gnanakaran, S.; Nussinov, R.; Garcia, A. E. (2006) Atomic-Lev el Description of Amyloid -Dimer Formation. J. Am. Chem. Soc. 128(7); 2158-2159. 22) Erlund, I. (2004) Review of the fla vonoids quercetin, hesperetin, and naringenin. Dietary sources, bioactivities, bioavailability, and epidemio logy. Nutrition Research, 24, 851-874. 23) Mandel, S., Amit, T., Reznichenko, L ., Weinreb, O., and Youdi m, M. (2006) Green tea catechins as brain-permeable, natural iron chelators-antioxidants for the treatment of neurodegenerative disorders. Nutr. Food Res, 50, 229-234. 24) Casetta, I., Govni, V., a nd Granieri E. (2005) Oxida tive stress, antioxidants and neurodegenerative diseases. Curr. Pharm. Des., 2005;11: 2033-52. 25) Furusawa, M., Tanaka, T., Ito, T., Nish ikawa, A., Yamazaki, N., Nakaya, K., Matsuura, N., Tsuchiya, H., Nagayama, M., and Iinuma, M. (2005) Antioxidant Activity of Hydroxyflavonoids. Journal of Health Science, 51(3), 376-378. 26) Chen, Z., Zhu, Y, Q., Wong, F, Y., Zhang, Z, and Chung, H. (1998) Stabilizing effect of ascorbic acid on green tea cat echins. J. Agric. Food Chem., 46, 2512-2516. 27) Cabrera, C., Gimenez, R., and Lopez, M. C. (2003) Determina tion of tea components with antioxidant activity J. Agric. Food Chem, 51 15, 4427-4435.
26 28) Zaveri, Nurulain. (2006) Green tea and its polyphenolic catechins: medicinal uses in cancer and noncancer applic ations. Life Sci., 78, 2073-2080. 29) Valcic, S., Muders, A., Jacobsen, E, N., Liebler, C, D., and Timmermann, N, B. (1999) Antioxidant Chemistry of Green Tea Ca techins. Identificati on of Products of the Reaction of (-)-Epigallo catechin Gallate with Peroxyl Ra dicals. Chem. Res. Toxicol., 12, 382-386. 30) Choi, Y., Jung, C., Lee, S., Bae, J., Baek, W., Suh, M., Park, J., Park, C., and Suh, S. (2001) The green tea polyphenol (-)-epigalloca techin gallate attenuates beta-amyloidinduced neurotoxicity in cultured hippocampal neurons. Life Sciences, 70, 603-614. 31) Rezai-Zadeh, K., Shytle, D., Sun, N., Mori, T., Hou, H., Jeanniton, D., Ehrhart, J., Townsend, K., Zeng, J., Morgan, D., Hardy, J., Town, T., and Tan J. (2005) Green Tea Epigallocatechin-3-Gallate (E GCG) Modulates Amyloid Precursor Protein Cleavage and Reduces Cerebral Amyloidosis in Alzheimer Transgenic Mice J. of Neuroscience 25, 38, 8807-8814. 32) Bocco, A., Cuvelier, M., Richard, H., and Berset, C. (1998) An tioxidant Activity and Phenolic Composition of Citrus Peel and Seed Extracts. J. Agric. Food Chem., 46, 21232129. 33) Croft, D, K. Annals NY Academy of Sci. (1998) The Chemistry and Biological Effects of Flavonoids and Phenolic Acids. 854, 435-441. 34) Burda, S. and Oleszek, W. (2001) An tioxidant and Antiradical Activities of Flavonoids. J. Agric. Food Chem., 49, 2774-2779.
27 35) Thompson, M. and Williams, C. R. (1976) Antioxidant Potential of Ecklonia cavaon Reactive Oxygen Species Scavenging, Metal Chelating, Reducing Power and Lipid Peroxidation Inhibition. Anal ytica Chimica Acta, 85, 375-381. 36)Asard, H., May, J,. and Smirnoff, N. V itamin C Functions and biochemistry in animals and plants. Garla nd Science: New York, 2004. 37) Tang, L., Li, M., Cao, P., Wang, F., Chang, W., Bach, S., Reinhard t, J., Ferandin, Y., Galons, H., Wan, Y., Gray, N., Meijer, L., Jia ng, T., Liang, D. (2005) Crystal Structure of Pyridoxal Kinase in Complex with Roscovitin e and Derivatives. J. Bio. Chem., 280, 35, 31220-31229. 38) Dolphin, D., Poulson, R., Avramovic, O. Vitamin B6 Pyridoxal Phosphate, Volume 1. 1986, John Wiley & Sons. 39) Yokochi, N., Morita, T., Yagi, T. ( 2003) Inhibition of Di phenolase Activity of Tyrosinase by Vitamin B6 Compounds. J. Agric. Food Chem., 51, 2733-2736. 40) Viola, R. and Hancock, R. (2005) Biosynthesis and Catabolism of L-Ascorbic Acid in Plants. J. Agric. Food Chem. 53, 5248-5257. 41) Saucier, T, C. and Wate rhouse, L, A. (1999) Synergetic Activity of Catechin and Other Antioxidants. J. Agric. Food Chem. 47, 4491-4494.
28 Chapter Two Blastula Protease-10 Peptide as Tyrosinase-like Mimic Introduction/ Rationale Blastula Protease 10 (BP10) is a mononuclear Zn-dependent endopeptidase that is involved in sea urchin embroyogenesis.1 The enzyme utilizes a structural motif (HE xx H xx G xx H) and a Tyr ligand following a distant Met turn to coordinate the Zn2+ ion. These conserved struct ures are found in nearly 30 different enzymes and are classified as metzinicins.2 The exact role of BP10 in embroyogenesis is still unknown. Furthermore, it is difficult to make comparisons to other members of the metzincins because each differs remarkably in localizat ion. In a recent study, the copper derivatives of BP10 was prepared and have been show n to be more hydrolytically active in comparison to that of the native Zn-derivative.1 The binding of Cu2+ to the His rich motif has alluded to the possibility of additional types of Cu-chemistry. For years, model complexes have b een prepared to characterize both intermediates and mechanisms for Type 3-Copper proteins, such as Tyrosinase.3,4 Tyrosinase is an enzyme found in both plants and animals, responsible for the synthesis of melanin. This enzyme is well studied partially due to its agricultural significance, specifically its role in the browning of food. These model complexes tend to be nitrogen rich and activity is usually show n in mixed organic/aqueous solvent.3 Only in the past few years have begun to use model pep tides to mimic enzymatic catalysis.5,6 The next chapter will concern the use of the metzincin motif from BP10 as tyrosinase mimic in aqueous media. Metal binding and mechanis tic information is alluded to by various kinetic experiments.
29 Experimental: Chemicals and Materials for Metal Titrations and Kinetics Assays: The BP10 peptide was synthesized and pur chased from the University of South Florida Peptide Center. The identity of the 21 amino acid peptide (GIVHE IGHAI GFHHE QSAPD R) was confirmed with a Bruker matrix-assisted laser desorption ionization MALDI time-of-flight mass spectrometer. The buffer used in all assays is 100 mM HEPES at pH 7, with small amount of ch lex resin to demetalize the solution. EDTA was used in cleaning glass/plastic ware pr ior to usage in order to prevent metal contamination. Deionized water of 18 M was obtained from a Milli Q system (Millipore, Bedford, MA) and used for all cleaning and for preparation of stocks solutions. CuSO4 and ZnSO4 were used for all experiments. All kinetic studies were run using a Varian CARY50 Bio-UV-Vi s spectrophotometer at 293 K. Peptide Preparation The molar absorptivity was determined by monitoring the absorbance of known concentrations of peptide dissolved in water at 280nm for phe nylalanine. Metal derivatives were prepared by the addition of a known concentration of metal to achieve a 1:1 ratio of metal to peptide. Fresh peptid e stocks were prepared and used within 24 hours. Metal Binding Apo-BP10 was diluted in 100 mM HEPES at pH 7.00 to a final concentration of 0.5 mM. The binding of Cu2+ was monitored by titrating metal into apo-BP10 and collecting the spectra after each additional of metal. Cu2+ binding was also determined
30 through oxidative activity of Cu2+-BP10 complex toward catechol. In 100 mM HEPES pH 7.00 buffer, 2mM catechol, and the 2 mM o-quinone indicator 3-methyl-2benzothiazolinone hydrazone hydrochloride mo nohydrate (MBTH), activities of various ratios of Cu:BP10 were monitored at 500nm for the o-quninone-MBTH complex (Figure 2-1). Additionally, Cu2+/Zn2+ at various ratios were titrat ed to BP10 and the oxidation of catechol (conditions same as Cu2+ titration) monitored. OH S N N NH3+ CH3 O O OH O S N N N CH3 OH OH Hydroxylation Oxidation Phenol Catechol MBTH o-quinone Figure 2-1: Scheme showing the bindi ng of o-qunione indicator 3-methyl-2benzothiazolinone hydrazone hydroc hloride monohydrate (MBTH)
31 Enzyme Kinetics The study of the effect of changing expe rimental conditions on the rate of an enzyme-catalyzed reaction is known as enzyme ki netics. In most studi es, the initial rate, Vo, varies almost linearly with substrate con centration, [S] is determined. At higher [S], Vo response is decreased, eventually being vi rtually unaffected by any addition of S. This seemingly constant rate is considered as the maximum velocity, Vmax. The reaction between the enzyme, E, and S, yields an ES complex, a necessity for the next step in enzymatic catalysis. k1E + S ES k2E + P k-1 When the enzyme is initially introduced to the substrate, the reaction quickly achieves a steady state, with the ES comple x remaining constant over time. The ES complex then breaks down to yield a product (P) and an E that is able to catalyze another reaction. The breakdown of the ES complex is used to determine Vo (Equation 2.1). [ES] k V2 o Equation 2.1 Experimentally it is difficult to determin e [ES], making it impor tant to consider alternative methods to determine Vo. Utilizing a steady-state assumption that states the [ES] complex is formed and broken-down at an equivalent rate, one can derive an equation that can determine Vo though the use of experimentally derived parameters. The rate of ES formation and breakdown can be define by equations (Equation 2.1, 2.2) [ES])[S] ] ([E k dt d[ES]t 1 Equation 2.2 [ES] k [ES] k dt d[ES] -2 1 Equation 2.3
32 Where [Et] is the total enzyme concentration (both in E and ES). Setting these equivalent and through some algebraic manipul ation to solve for [ES], yields Equation 2.4. 1 1 2 tk ) k (k [S] ][S] [E [ES] Equation 2.4 By substituting equation 2.4 into equation 2.1, one can express the equation in terms of Vo (Equation 2.5). 1 1 2 t 2 ok ) k (k [S] ][S] [E k V Equation 2.5 Because Vmax is defined as the maximum velocity attained after the enzyme is saturated (Equation 2.6), the equation to solve for Vo can further be simplified (Equation 2.7) ] [E k Vt 2 max Equation 2.6 1 1 2 max ok ) k (k [S] [S] V V Equation 2.7 Another parameter of particular importa nce is the Michaelis-Menten constant (Km). This is usually defined as the substrate concentration that has a rate equal to half the Vmax. Km is solved to give Equation 2.8. 1 1 2 mk k k K Equation 2.8 Substituting this equation in equation 2.7 yields what is known as the MichaelisMenten equation (Equation 2.9) a nd is depicted in figure 2-2.
33 [S] K [S] V Vm max o Equation 2.9 Depending on the rate limiting step, specifically when k2 << k-1, Km can be used to represent the affinity of E to S in th e ES complex. When this condition holds, Km is defined as the dissociation constant (Kd) (Equation 2.10), of the ES complex. 1 1 dk k K Equation 2.10 Since enzymes can react in fashions th at the rate limiting step is not the degradation of ES, the firs t-order rate constant kcat is often used to report rates in terms of turnover per time (Equation 2.11). [E] V kmax cat Equation 2.11 [S] Vo 1/2 V max K m V max Figure 2-2: Mich aelis-Menten plot.
34 Furthermore, to compare enzymes the second order rate constant kcat/Km (specificity constant) is used to desc ribe the conversion of E + S to E + P. Another common technique to determine kinetic parameters is through the use of a double-reciprocal or Lineweaver-Burk plot (Equation 2.12, Figure2-3). max max m oV 1 [S]V K V 1 Equation 2.12 The Lineweaver-Burk plot is particularly useful to distinguis h types of inhibition patterns, including competitive, noncompetitive, uncompetitive, and mixed-type inhibition. A competitive competes for the acti ve site of an enzyme with the substrate. This direct competition of the inhibitor (I ) can be overwhelmed by increasing amounts of x 1/[S] 1/Vo 1/K m 1/V max Slope = K m / V max Figure 2-3: Line weaver-Burk plot.
35 S. This type of inhibiti on has a trend of increasing Km and relativity constant Vmax. Competitive inhibition is depicted by the sc heme, equations, and plot in Figure 2-4. k1E + S ES k2E + P I + E Ki 1 Vo = Km Vmax 1 [S] ( ) Ki [I] 1 + + 1 Vmax k-1 1/[S] -1001020304050 1/V o 0 10 20 30 40 50 Increasing [I] max max1 ) ] [ 1 ( ] [ 1 1 V K I S V K Vi m o Figure 2.4: Graphical, schematic, and equations for competitive inhibition Another type of inhibition, considered nonc ompetitive, is when the inhibitor binds both E and the ES. This type of inhib ition usually has the trend of increasing Vmax and constant Km. This type of inhibition is show n by the following Lineweaver-Burk plot trend and equations in figure (2-5).
36 k 1E + S ES k2E + P I + EI Ki + S I + ESI V = Vmax [S] ([S] + Km) ( 1+ [I]/Ki) k-1 Kis 1/[S] -1001020304050 1/Vo 0 10 20 30 40 50 Increasing [I] Figure 2.5: Graphical, schematic, a nd equations for noncompetitive inhibition A third type of inhibition is a mix between competitive and non-competitive, appropriately named mixed type Mixed type inhibition is the same equilibrium as noncompetitive, with inhibitor binding at di fferent affinities to both the E and ES complex. (Figure 2.6)
37 k1E + S ES k2E + P Vmax(app) Km(app) = Vmax K m [I] Ki Kis 1 + Vmax(app = Vmax [I] 1 + I + EI Ki + S I + Kis ESI k-1 1/[S] -1001020304050 1/Vo 0 10 20 30 40 50 Increasing [I] Figure 2.6: Graphical, schematic, and equations for mixed-type inhibition. The forth type of inhibition which is when the inhibitor binds only to the ES complex. An inhibitor is considered to be uncompetitive when it influences the rate by binding to a location other then substrate bindi ng site. The binding to the ES complex is associated with decreasing Km and Vmax. (Figure 2.7)
38 k1k -1E + S ES k2E + P = K m [I] Ki 1 + Vmax + I + Ki ESI Vo [S] [S] ( ) 1/[S] -1001020304050 1/Vo 0 10 20 30 40 50 Increasing [I] Figure 2.7: Graphical, schematic, a nd equations for uncompetitive inhibition
39 Catechol/Phenol oxidation Assays Using a constant Cu-BP10 concentration (210M) with a 1:1 Cu to peptide ratio, various substrate concentrations were assayed. The final volume of each assay is 1 mL at pH 7.00 100 mM HEPES and 298 K. The concentr ation of MBTH was kept in proportion with substrate concentration. Catechol was varied 0.05-1.2 mM and the MBTH-oquinone product was monitored at 500 nm for 35 mins (Figure 2-8A) The rates were determined by the change in absorbance over time (Figure 2-8B). A similar assay was constructed for phenol with concentrations ranging from 0.2-3.2 mM and was also monitored at 500 nm for o-quinone production.
40 Hydrogen peroxide (H2O2) titration was perfomed with fixed catalyst and saturating amount of substrate. Th e conditions were similar to non-H2O2 assays described above. H2O2 varied from 0.25mM-12mM and the catecho l/phenol-MBTH product ( = 32,500 M-1 cm-1) was monitored at 500 nm. Additionally, experiments were nm 440460480500520540560580600 Abs (A) Time (mins) 0123 Abs 500nm (B) Figure 2-8: (A) The production of o-qui none from catechol monitored by the increase in absorption as a result of th e formation of its adduct with 3-methyl-2benzothiazolinone hydrazone hydrochlorid e monohydrate (MBTH). (B) Monitoring the increase in absorption at 500 nm for catechol oxida tion to obtain the rate.
41 preformed that varied catechol conc entration at a fixed catalyst and H2O2 concentration. The assays preformed had a [H2O2] fixed at 0.25, 0.75, 1.5, 3.0, or 6.0 mM. Deuteratedphenol (d-phenol) experime nts were performed under the same conditions as described above. Using 10 M Cu2+-BP10 and varying d-phenol from .43.2 mM the absorbance was monitored at 500 nm. Furthermore, under saturating conditions of H2O2 (20 mM), d-phenol was titrated. Inhibition Experiments Conditions for inhibition experi ments consisted of 0.5-2 M Cu2+-BP10, pH 7.00, 100 mM HEPES buffer, 293 K, and 1 ml total volume. To obtain the Dixon plot, kojic acid was titrated into assays containing fixed catechol and MBTH concentrations (0.3mM). Kojic acid concentration varied from 0.025 0.8 mM. Catechol oxidation was then monitored at various concentrations of kojic acid (0.25, 0.05, and 0.1 mM) at 500 nm. Cyanide inhibition was monitored under similar conditions to kojic acid inhibition. A dixion plot wa s obtained by titrating cyanide into a fixed amount of catechol (0.3mM) and monitoring for the form ation of the o-qunione. Catechol oxidation was then monitored at various concentrations of cyanid e (0.002,0.005, 0.0035, mM) to obtain the Lineweaver-Burk plot A Dixon plot was then obt ained that kept catechol, MBTH, catalyst, and H2O2 (0.7 mM) constant, while titrating cyanide. Assays were then performed at 0, 0.015 and 0.03 mM cyanide, while varying H2O2 from 0.125-10 mM. A third inhibition experiment was preformed that varied catechol at various cyanide concentrations (0, 0.015, 0.03 mM) while keeping H2O2 under saturating condition (8 mM).
42 Results and Discussion Metal Binding To examine the metal-coordination envi ronment, the electronic spectrum of Cu2+BP10 was obtained (Figure2.5). Upon the addition of Cu2+, there is a d-d transition with a max of 610 nm. The spectrum is analogous to type-2 copper centers and distinct from aqueous Cu2+ absorbance at 820 nm.6 Furthermore the spectrum is comparable to published Cu2+-bound His-rich peptides.6 In order to gain further insight into the metalcentered redox chemistry, activity was also used to confirm the Cu2+:BP10 stoichiometry. By measuring the activity at various equivalents of Cu2+, the resulting data saturates around 1:1 ligand-to-metal ratio (Figure 2.6). The data reveals a sigmoidal pattern which is fit to the Hill equation yieldi ng a Hill coefficient of 2.87. In general, a Hill coefficient greater then unity indicate s a positive cooperatively. For comparsion purposes, the coefficient for Cu2+ binding to BP10 is equivalent to that of O2 binding to hemoglobin with a Hill Coefficient of 2.8. To gain furt her insight into the metal-center, diluting Cu2+BP10 with Zn2+ would effectively silence the redox chemistry. If the catalysis is carried out by a mononuclear Cu2+-center, the Zn2+ should replace the Cu2+ and result in a noncooperative nearly linear binding. Figure 2.6 indicates a sigm odal relationship, yielding a Hill coefficient of 1.76. This suggests the possible presence of a cooperative Cu2+ binding to form a Type-3 copper center dur ing the catalysis of catechol, corroborating with the result in direct Cu2+ binding (Figure 2.,6 Top)
43 wavelength/nm 400500600700800900 Abs 0.00 0.01 0.02 0.03 0.04 Figure 2.9: Electronic spectra of Cu2+-BP10 with 1 equivalent of Cu2+-BP10. (100 mM HEPES buffer at pH 7.0, 0.5 mM BP10)
44 Cu2+ Equiv. 0.00.20.40.60.81.01.21.41.6 Rate (mM/s) 5.0e-6 1.0e-5 1.5e-5 2.0e-5 2.5e-5 3.0e-5 3.5e-5 nCu/(nCu + nZn) 0.00.20.40.60.81.0 Rate (mM/s) 1e-5 2e-5 3e-5 4e-5 5e-5 6e-5 Figure 2.10: (Top) Cu2+ titration to BP10 monitored with the oxidation of catechol. Fit to Hill equation, which yields a Hill coefficient of 2.86 0.18. (Bottom) Oxidative activity of Cu2+-BP10 toward the oxidation of catechol as a function of the mole fraction of Cu2+ at a constant total concentration of Cu2+ and Zn2+. Fit to Hill equati on, which yield Hill coefficient of 1.76 0.17. (B oth assays contained [BP10] = 6 M, [MBTH] = [catechol] = 2mM, 100 mM HEPES buffer pH 7.0, 293 K)
45 Catechol/Phenol Oxidation The oxidation of catechol to o-quinone is a 2-el ectron transfer that favors the presences of a dinuclear Cu2+ center. Studies concerning tyrosina se and catechol oxidase have shown that once the dinuclear center is in the met form, catechol can readily bind and be oxidized to its quinone product.7 In the presence of O2, micromolar amounts of Cu2+BP10 can readily oxidize catechol and is satu rated at mM amounts of substrate (Figure 2.7), yielding a kcat = 4.06 s-1, Km = 0.254 mM, and a significant second-order rate constant of 1.60 x 104 M-1 s-1. In terms of first order rate constant, Cu2+-BP10 is 8.57 x 106 fold higher than the autooxidation of catechol (ko = 4.74 x 10-7 s-1) and 7.5 fold higher then another catechol oxi dizing peptide mimic (0.531 s-1).6
46 [Catechol] mM 0.00.20.40.60.81.01.21.4 Rate (mM/s) 2.0e-6 4.0e-6 6.0e-6 8.0e-6 1.0e-5 1.2e-5 1.4e-5 1.6e-5 Km (mM) 0.254 .020 Vmax (mM/s) (1.63 .04) x 10-5 kcat (s-1) 4.06 kcat/Km (mM-1 s-1) 16.0 Figure 2.11: Cu2+-BP10 oxidation of catechol at pH 7.00 and 293K. [Cu2+-BP10] kept constant at 4 M. Table includes kinetic parameters.
47 To gain further insight into the mechanism of Cu2+-BP10, H2O2 was titrated into the complex with saturating amounts of cat echol (Figure2.8). The data showed a significant increase in rate and was saturated after mM amounts of H2O2. The saturation kinetics observed for both catechol and H2O2 implies a possible bisubstrate mechnaism, wherein both can bind to the metal active center. To obtain apparent and [H2O2] mM 0246810 Rate (mM/s) 2.0e-5 4.0e-5 6.0e-5 8.0e-5 1.0e-4 1.2e-4 1.4e-4 1.6e-4 Km (mM) .967 .188 Vmax (mM/s) 1.27x10-4 6.12x10-6 kcat (s-1) 31.8 kcat/Km (mM-1 s-1) 32.8 Figure 2.12 The effect of H2O2 on Cu2+-BP10 oxidation of catechol in the presence of saturating catechol (1.5mM) at pH 7.00 and 293K. [Cu2+-BP10] kept constant at 4 M.Tableincludeskineticparameters.
48 intensic dissociation constants for catechol and H2O2, the rates at varying amounts of [H2O2] holding [catechol] constant and vise versa were determined (Figure 2.9). The data could be fitted to a two-substrate rand om-binding equilibrium shown below. k2E + P + EA KiA + B + EAB A A KiB KA KB E + B EB ] O [H K K V K [Catechol] ] O [H V K V 1 V Catechol2 2 int(C) app(C) max app(C) 2 2 max app(H) max o Figure 2-13: Random bisubsubstrat e equation and equilibrium. In the equation, Kapp(H) is the apparent affinity constant for H2O2, Kapp(C) is the apparent affinity consta nt for catechol, and Kint(C) is the intrinsic affinity constant for catechol. From the Hanes analysis, a secondary plot of the slope (1/Vmax) and the yintercept (Kapp(Substrate)/Vmax) verus 1/[Substrate] is obtai ned (Figure 2.10). From the slopes and y-intercepts of these secondary pl ots, the apparent and intensic dissociation constants can be obtained. Using the ratios of Kapp/Kint the effect of the binding of one substrate on the other can be measured. If th e ratio is above 1, then the binding of one ligand decreases the affinity for the other, be low one represents an increased affinity, and equal to 1 indicates no effect on one a nother. From the results obtained
49 [H 2 O 2 ] mM 0246 k cat s -1 0.005 0.010 0.015 0.020 0.025 0.030 0.035 [Catechol] mM 0.00.20.40.60.81.01.21.41.6 Rate (mM/s) 2e-5 4e-5 6e-5 8e-5 1e-4 Figure 2.14: (Top Plot) The e ffect of the concentration of H2O2 on the first-order rate constant kcat toward the Cu2+-BP10 oxidation of cat echol. (Bottom Plot) 0 ( ), 0.25 ( ), 0.75 ( ), 1.5 ( ), 3.0 ( ), and 6 mM ( ) H2O2 effect on the rate of catechol oxidation. Conditions at pH 7.0 and 293 K, [Cu2+-BP10] = 2 M.
50 Kapp(C)/Kint(C) =0.752, while Kapp(H)/Kint(H) = 1.04. From the Hanes analysis, catechol seems to have no effect on H2O2 binding, while H2O2 increases the affinity for catechol slightly. Although these results provide insight into the Cu2+-BP10 mechanism, al one they provide insufficient evidence to concl ude the sequencal binding. In addition to catechol oxidation, Cu2+-BP10 was shown to hydroxylate and oxidize phenol to the o-qunione product. Phenol hydroxylation is often times challenging for metal-centered chemistry because it is a spin-forbidden process, inserting the triplet O2 into the singlet C-H bond. Furthermore, the aerobic hydroxylation/oxidation of phenol is relativity slow (k0 = 4.60 x 10-8 s-1).6 Cu2+-BP10 was shown to significantly enhance the tyrosi nase-like hydroxylation activity by 8.57 x 103 times (kcat = 3.94 x 10-4 s-1). The rate compared to catechol oxidation is significantly reduced by around 1 x 105 times, believed to be in part due to the difficult hydroxylation step. To further inquire if in fact the rate determining st ep is the hydroxylation, d-phenol was used as a substrate. The rate of the reaction remained relatively unchanged, with a kinetic isotope effect of only 1.27. This result indicates that hydroxylation is most likely not the rate determining st ep of the reaction. [H2O2] mM Km (mM) Vmax (mM/s) kcat (s-1) kcat/Km (M-1 s-1) 0 0.25 0.02 (1.63 0.04) x 10-5 4.06 16.0 x 103 0.25 0.40 0.06 (3.97 0.20) x 10-5 19.9 49.4 x 103 0.75 0.29 0.01 (5.47 0.08) x 10-5 27.4 93.5 x 103 1.5 0.41 0.04 (8.56 0.26) x 10-5 42.8 103 x 103 3.0 0.27 0.03 (9.40 0.35) x 10-5 47.0 173 x 103 6.0 0.39 0.08 (1.19 0.78) x 10-4 59.5 149 x 103 Table 2.1: Kinetic parameters for H2O2 effect on Cu2+-BP10 oxidation of catechol.
51 [Catechol] mM 0.00.20.40.60.81.01.21.41.6 [Catechol]/Rate (s) 0 2e+4 4e+4 6e+4 8e+4 1e+5 1/[H2O2] mM-1 012345 y-intercept; slope 0.0 5.0e+3 1.0e+4 1.5e+4 2.0e+4 2.5e+4 3.0e+4 [H2O2] mM 01234567 [H2O2]/Rate (s) 0.0 5.0e+4 1.0e+5 1.5e+5 2.0e+5 2.5e+5 1/[Catechol ] (mM-1) 024681012 Y-intercept; Slope 0.0 5.0e+3 1.0e+4 1.5e+4 2.0e+4 2.5e+4 3.0e+4 3.5e+4 4.0e+4 Km (C) (mM) 0.427 Kapp (C) (mM) 0.321 Km (H) (mM) 0.535 Kapp (H) (mM) 0.558 Kapp (H) / Km (H) 1.04 Kapp (C) / Km (C) 0.752 Figure 2.15: (Top) Hanes analysis of vari ous [catechol] and secondary plot (slope yintercept ). (Bottom) Hanes analysis of various [H2O2] and secondary plot (slope y-intercept ). Table includes apparent and intrin sic affinity constants for catechol and H2O2.
52 [Phenol] ; [d-Phenol] mM 0.00.51.01.52.02.53.0 Rate (mM/s) 5.0e-7 1.0e-6 1.5e-6 2.0e-6 2.5e-6 3.0e-6 Phenol d-Phenol Km (mM) 1.67 .218 1.40 .176 Vmax (mM/s) (3.9 0.2) x 10-6 (3.1 0.2) x10-6 kcat (s-1) 3.94x10-4 3.05x10-4 kcat/Km (mM-1 s-1) 2.36x10-4 2.18x10-4 Figure 2-16: Cu2+-BP10 hydroxylation/oxidation of phenol ( ) and d-phenol ( ) without H2O2. Table includes kinetic parameters.
53 Inhibition The results thus far have indicated that both H2O2 and catechol/phenol are substrates for Cu2+-BP10. Further detailed mechanistic inferences can be made by the use of oxygen and catechol mimics as inhibitors. A popular compet itive inhibitor for Type-III Cu-centers is kojic acid.8 As seen in Figure 2-12, kojic acid shows to be a competitive inhibitor for catechol oxidation by Cu2+-BP10. The low Ki indicates the inhibitor has tight binding and is relatively specific for the catalyst. Kojic acid inhibition further supports the notion of the presence of a dinuc lear center and that ca techol binds to the same location as kojic acid. To gain insight into the role and bind ing of the oxygen species cyanide was used as an inhibition. Cyanide is a wellknown oxygen mimic that has been used to characterize O2 binding sites. In figure 2-13, cyan ide is used in the presence of atmospheric O2 while titrating catechol. The mixed type inhibition and near equal Ki and Kis indicate cyanide binds to both the E and ES complex with similar affinity. Since the concentration of O2 in solution is unknown and may not be at saturating conditions, the type of inhibition for this assay reveal s only the possible presence competitive and uncompetitive inhibition and that cyanide can bi nd to the E and/or ES complex. Another cyanide inhibition assay checked the inhibition in the presence of saturating conditions of catechol while titrating H2O2 (Figure 2-14). The result s reveal a clear noncompetitive pattern between cyanide and H2O2. The inhibitor in noncompetitive inhibition binds both the E and the ES complex. Be ing that catechol is at sa turating conditions and bound first to E, cyanide could possibly serve as a reducing agent stabilizing and blocking Cu+ and thus preventing O2 from binding. The third cyanid e inhibition experiment involved H2O2
54 [Kojic Acid] mM 0.00.20.40.60.8 Rate (mM/s) 2e-6 4e-6 6e-6 8e-6 1/[Catechol] (mM-1) 0510152025 1/rate (mM-1 s) 0.0 2.0e+5 4.0e+5 6.0e+5 8.0e+5 1.0e+6 1.2e+6 1.4e+6 [Kojic Acid] mM Km (mM) Vmax (mM/S) 0.00 0.202 .016 (1.30 0.03 ) x 10-5 2.50e-2 0.344 .013 (1.39 0.02) x 10-5 5.00e-2 0.435 .041 (1.39 0.05) x10-5 1.00e-1 0.534 .074 (1.28 0.07) x 10-5 Ki= 0.043 mM Figure 2-17: Kojic Acid Inhibition(Top) Ko jic acid titration into constant [catechol] and [Cu2+-BP10]. (Bottom) Lineweaver-Burk plot ti trating catechol at different [kojic acid] ( 0 mM, ; 0.025 mM, ; 0.05 mM, ; 0.10 mM, ) (Table) The effects of [kojic acid] on Vmax and Km, in addition to the Ki for competitive inhibition.
55 [Cyanide] M 05101520253035 Rate (mM/s) 0 1e-6 2e-6 3e-6 4e-6 5e-6 6e-6 7e-6 8e-6 1/[Catechol] mM-1 0510152025 1/rate (mM-1 s) 0.0 2.0e+5 4.0e+5 6.0e+5 8.0e+5 1.0e+6 1.2e+6 1.4e+6 1.6e+6 [Cyanide] mM Km (mM) Vmax (mM/s) 0.00 0.132 .008 (8.96 0.16) x 10-6 5.0 x 10-4 0.166 .005 (8.59 0.08) x 10-6 2.0 x10-3 0.211 .034 (6.22 0.33) x 10-6 3.5 x 10-3 0.223 .035 (4.06 0.21) x 10-6 Ki = (1.4 1.7) x10-3 mM | Kis = (6.3 4.7) x10-3 mM Figure 2-18: Cyanide Inhibition in the presence of O2. (Top) Cyanide titration into constant [catechol] and [Cu2+-BP10]. (Bottom) Lineweav er-Burk plot titrating catechol at different [cyanide] ( 0 mM 0.5M 2.0 M 3.5 M ) (Table) The effects of [cyanide] on Vmax and Km, in addition to the Ki (Interaction with free E) and Kis (interaction with the ES complex) for mixed type inhibition.
56 [Cyanide] mM 0.0000.0050.0100.0150.0200.0250.0300.035 Rate (mM/s) 0 1e-6 2e-6 3e-6 4e-6 5e-6 6e-6 7e-6 8e-6 1/[H2O2] mM-1 -2-101234 1/Rate (mM-1 s) 0 2e+5 4e+5 6e+5 8e+5 1e+6 [Inhibitor] mM Km (mM) Vmax (mM/s) 0.0 0.496 .052 (6.31 0.19) x 10-6 1.5 x 10-3 0.522 .034 (3.57 0.08) x 10-6 3.0 x 10-3 0.764 .137 (3.17 0.23) x 10-6 Ki= 3.03x10-3 mM | Kis= 1.45x10-3 mM Figure 2-19: Cyanide Inhibi tion in the presence of H2O2. (Top) Titrating cyanide into fixed [Catechol], [Cu2+-BP10], and [H2O2]=.7mM. (Bottom) Lineweaver-Burk plot titrating H2O2 at different [cyanide] ( 0 mM, ; 1.5 M, ; 3.0 M, ) while keeping [catechol] at saturating conditions. (Table) The effects of [cyanide] on Vmax and Km, in addition to the Ki for noncompetitive inhibition.
57 binding. The third cyanide inhibition experiment involved H2O2 at saturating conditions while titrating catechol (Figure 2-15). Th e results clearly represents uncompetitive inhibition of cyanide against catechol. An uncompetitive inhibitor binds only to the ES complex. Since cyanide is considered an oxygen mimic, the results suggest that oxygen (cyanide) would bind to the active center after catechol is bound to form the Cu2+-BP10catechol complex. 1/[catechol] m-1 -20246 1/rate (mM-1 s) 0 2e+5 4e+5 6e+5 8e+5 1e+6 [Inhibitor] mM Km (mM) Vmax (mM/s) 0.0 1.06 0.28 (1.24 0.16) x10-5 1.5 x 10-3 .590 0.085 (5.19 0.94) x 10-6 3.0 x 10-3 .375 0.053 3.27 0.15) x10-6 Ki = 1.64 x 10-3 mM Figure 2-20: Cyanide Inhibi tion in the presence of H2O2. Lineweaver-Burk plot titrating catechol at di fferent [cyanide] ( 0 mM 1.5 M 3.0 M ) while keeping [H2O2] at saturating conditions (8mM). (T able) The effects of [cyanide] on Vmax and K m in addition to the K i
58 Closing Remarks The metzinicin motif found in BP10 has shown to bind Cu2+ and form a Type-III Cu-center. The complex is relatively activ e in comparison to the background rate of catechol and phenol oxidation. Furthermore, it shows that H2O2 can enhance the reaction and serves as the second substrate in a bis ubstrate reaction. From the Hanes analysis, catechol seems to have no effect on H2O2 binding, while H2O2 increases the affinity for catechol slightly. The catechol binding to the free E was confirmed by the kojic acid inhibition. Cyanide inhibitions confirmed that oxygen is involved in the reaction and that cyanide binds only after catechol binding.
59 Reference 1) Da Silva, GFZ. Reuille, L, R., Ming, L., a nd Livingston, T. (2006) Overexpression and Mechanistic Characterization of Blastula Prot ease 10, a Metalloprot ease Involved in Sea Urchin Embryogenesis and Development. J. Biol. Chem., 281, 16, 10737-10744. 2) Bode, W., Gomis-Rueth, FX., and Stoeckler, W. (1993) Astacins, serralysins, snake venom and matrix metalloproteinases exhi bit identical zinc-binding environments (HEXXHXXGXXH and Met-turn) a nd topologies and should be grouped into a common family, the metzincins, FEBS Letters, 331, 134-140. 3) Granata, E., Monzani, and Casella, L. (2004) Mechanistic insight into the catechol oxidase activity by a biomimetic dinuclear c opper complex. J. Biol. Inorg. Chem. 9, 903913. 4) Mahadevan, W., Gebbink, RK., and St ack, TDP. Curr. Opin. Chem. Bio. (2000) Biomimetic modeling of copper oxidase reactivity. 4, 228-234. 5) Casolaro, M., Chelli, M., Ginanneschi, M., Laschi, F., Messor i, L., Muniz-Miranda, M., Papini, AM., Kowalik-Jankowska, T., a nd Kozlowski, H. (2002) Spectroscopic and potentiometric study of the SOD mimic syst em copper(II)/acetyl-L-histidylglycyl-Lhistidylglycine. J. Inor g. Biochem. 89, 181-90. 6) Da Silva, GFZ., Tay, WM., and Ming, L. (2005) Catechol Oxidase-like Oxidation Chemistry of the 1-20 abd 1-16 Fragme nts of Alzheimers Disease-Related -Amyloid Peptide: Their Structure-Activity Correlation an d the Fate of Hydrogen Peroxide. J. Bio. Chem., 280, 16601-16609.
60 7) Soloman, I, E., Sundaram, M, U., and M achonkin, E, T. (1996) Multicopper Oxidases and Oxygenases Chem. Rev., 96, 2563-2605. 8) Chen, J., Cheng-I Wei, C., Marshall, M. (1991) Inhibition Mech anism of Kojic Acid on Polyphenol Oxidase? J. Agric. Food Chem., 39, 18971901.
61 Chapter Three Alzheimers Disease and Natural Antioxidants Introduction/ Rationale Of neurodegenerative diseases, the most pr evalent is Alzheimers disease (AD). Although the past decade has made significant progr ess on the cause of the disease, it still remains somewhat of a mystery. Of the many hypotheses proposed, the common link seems to be amyloid -peptide (A ).1 This short peptide varies in length following secreatase cleavage of the am yloid precursor protein (APP).1 In general, shorter more soluble fragments are considered to be nonamyloidgenic, while longer hydrophobic fragments are considered the cause or effect of AD.1 Along with a microtubule stabilizing tau protein, longer fragments of A have shown to accumulate, forming plaques in the brain.1 Studies have shown these plaques to be responsible for alterations in normal brain function, such as abnormal Ca2+ homeostasis and production of H2O2.1,2,3 Furthermore, postmortem studies have revealed the presence of redox active metal (e.g. Cu2+) present in the plaques.4 The presence of this seemingly misguided metal has fueled the hypothesis of reactive oxygen species (ROS ) as a major component of neuronal cell loss. In addition, studies have shown A to bind metal with a relativity high affinity within the first 14 amino acids of the peptide.5 This study presents soluble fragments of Cu2+-bound A as highly redox active complexes. Compassions between fragments of A containing the amino acids believed to start dimerization will be examined.6 This will include the effect of ROS on the redox activity of Cu2+-A toward the neurotransmitter dopamine. In addition, natural antioxidants (e.g. flavonoids and vitamins) w ill be used to inhibit this AD-related redox
62 chemistry. This will allow for further infancies on the possible beneficial effect of numerous antioxidants and the identification of structural moieties that enhance the overall antioxidant activity. Cu2+OH Cu2+ O H OH Cu2+OH O Cu2+ O H+ O O Cu+OH Cu+ O2 H+ Cu3+O O2Cu3+ Cu2+O O Cu2+ Cu2+ O Cu2+ O Cu2+O O Cu2+ O O O O 2 H2O Catechol 2H+ 3H+ + H2O H2O2 H2O2 2H+ 2H+ H2O2 2H+ 2 eFigure (3-1) Purposed mechanis m for polyphenol oxidation by Cu2+-A .7
63 Experimental Chemicals and Materials for Metal Titrations and Kinetics Assays The A peptides (16 and 20 amino acid) were synthesized and purchased from the University of South Florida Peptide Ce nter. The identity of the peptides (DAEFR5HDSGY10EVHHQ15KLVFF20 and DAEFR5HDSGY10EVHHQ15K) were confirmed with a Bruker matrix-assisted laser desorption ioni zation time-of-flight (MALDI-TOF) mass spectrometer. The buffer us ed in all assays is 100 mM HEPES at pH 7.4 or 7.0, with small amount of chlex resin to demetalize the solution. EDTA was used in cleaning glass/plastic ware prio r to usage, in order to prevent metal contamination. Deionized water of 18 M was obtained from a Milli Q system (Millipore, Bedford, MA) and used for a ll cleaning and for preparation of stocks solutions. CuSO4 and CaCl2 were used for all experiments. All kinetic studies were run using a Varian CARY50 Bio-UV-Vis spectrophotometer. Peptide Preparation The molar absorptivity was determined by monitoring the absorbance of known concentrations of peptide dissolved in wa ter at 280nm for the aromatic amino acids. Metal derivatives were prepared by the a ddition of a known concen tration of metal to achieve a 1:1 metal to peptide ratio. Since A tends to coagulate, fresh peptide stocks were prepared and used within 24 hours. Dopamine and Flavonoid Oxidation assays Using a constant Cu2+-A concentration (1-6M) with a 1:1 Cu-to-peptide ratio, various substrate concentrations were assayed. The final volume of each assay is 1 mL at
64 pH 7.4 100mM HEPES and 298 K. The concentr ation of MBTH was kept in proportion with substrate concentration. Dopamine were varied from 0.1-2.5 mM and the MBTH-oquinone product was monitored at 510 nm for 3-5 mins. Similar assays were constructed for epicatechin (EC), epigallocatechin galla te (EGCG), and epigallocatehin (EGC) and were monitored at their respective max (460nm 465nm, and 460 nm). Hydrogen peroxide (H2O2) titration were performe d with fixed catalyst and saturating conditions of substrate. The conditions were similar to non-H2O2 assays described above. H2O2 varied and the dopamine/EC/ EGC/EGCG-MBTH product was monitored at their respective absorbencies Additionally, experiments were preformed that varied substrate at a fixed catalyst and H2O2 concentration. These data were then fitted to the Hanes analysis to determine a pparent and intrinsic dissociation constants. Molar Absorptivity The Molar Absorptivity ( ) was calculated by oxidizi ng a known concentration of substrate with tyrosinase with excess MBTH at the pH 7.4 100mM HEPES buffer. The for EGCG was found by the combination of value for EGC and gallic acid. Inhibition Experiments Conditions for inhibition expe riments consisted of M Cu2+-A pH 7.4 or 7.0 HEPES 100 mM buffer, 293 K, and 1 ml tota l volume. The inhibitors used were Substrates (pH 7.4) Dopamine EC EGC EGCG (M-1 cm-1) 10095 10040 7159 7665 Wavelength (nm) 510 460 460 465 Table 3-1: Molar Absorptivity values for neurotransmitter and flavonoids
65 quercetin, fisetin, taxifolin, ascorbic acid, and pyridoxamine. For all inhibitors a Dixon plot was obtained by titrating inhibition into a fixed concen trations of dopamine, MBTH, and Cu2+-A Then oxidation rates at different [dopa mine] were determined in a fixed [I] and [Cu2+-A ] to obtain the Lineweaver-Burk plots. Inhibition constants were determined from inhibition equations from Chapter 2. Attenuation of inhibition wa s monitored by titrating Ca2+ into a fixed concentration of fisetin, dopamine, and Cu2+-A Oxidation rate of dopamine was then determined at fixed concentration of fisetin, Cu2+-A and Ca2+to obtain kinetic parameters.
66 Results and Discussion Green Tea Dopamine is a catechol containing neur otransmitter found extensively throughout the body. Like other neurotransmitters, dopamine is used to amplify and regulate signals to dopamine receptors. An alteration in levels of dopamine (e.g. oxidation) is in general a hallmark of several neur odegenerative diseases.7 Alzheimers disease (AD) is associated with degradation of normal brain func tion which includes altered levels of neurotransmitters, influx of Ca2+, and accumulation of protein fragments.1,2 The results in figure 3-2 and 3-3 indicated Cu2+-A 1-16 and Cu2+-A 1-20 significantly accelerate aerobic oxidation of dopamine in terms of kcat relative to auto-oxidation rate constant ko = 1.59 x 10-8. Furthermore, the additional 4 amino acids of Cu2+-A 1-20 seem to have a negligible effect on dopamine oxidation. Through metal ion reduction, reports have indicated the production of H2O2 by metallo-A .2 The results in figure (3-5) and (3-6) indicated that H2O2 significantly increases th e rate of oxidation of dopa mine. The oxidation rate dependent on H2O2 eventually plateaus, concluding H2O2 binds Cu2+-A and is turned over. Since both dopamine and H2O2 are considered substrates for Cu2+-A 1-16 and Cu2+A 1-20 the data can be fitted to a bisubstr ate random-binding equation to obtain both apparent and intrinsic dissociation constants Kapp and Km. (Table 3-2). The oxidation and generation of ROS in AD brains have suggested possible benefit from the consumption of f oods with high antioxidant content.9 A class of compounds reported to have antioxidant, anti radical, and influence on APP processing
67 are the green t ea catechins (GTC).10,11 The three GTCs were shown to be substrates for both Cu2+-A 1-16 and Cu2+-A 1-20 (Figures 3-2 3-4). [Substrate] mM 0.00.51.01.52.02.5 Rate (mM/s) 5.0e-6 1.0e-5 1.5e-5 2.0e-5 2.5e-5 3.0e-5 Cu2+-A 16 Dopamine EC EGCG Km (mM) 0.269 0.033 0.830 0.095 0.215 0.012 Vmax (mM/s) (1.45 0.06) x 10-5 (1.08 0.05) x10-5 (2.73 0.05) x 10-5 kcat (s-1) 4.83 x 10-3 3.60x10-3 9.10x10-3 kcat / Km (mM-1 s-1) 0.0180 4.34x10-3 .0423 Figure 3-2: Saturation kinetic profile for the oxidation of dopamine ( ), epicatechin (EC) ( ), and epigallocatechin gallate (EGCG) ( ) using Cu2+-A 16 (3 M) at 100 mM HEPES pH 7.4, 298K. Table includes kine tic parameters for dopamine, EC, and EGCG oxidation by Cu2+-A 16.
68 [Substrate] mM 0.00.20.40.60.81.01.21.41.6 Rate (mM/s) 2e-5 4e-5 6e-5 8e-5 1e-4 Cu2+-A 20 Dopamine EC EGCG Km (mM) 0.214 .050 0.302 0.016 0.310 0.053 Vmax (mM/s) (3.26 0.22) x 10-5 (2.66 0.05) x10-5 (9.52 0.52) x 10-5 kcat (s-1) 4.66x10-3 3.80x10-3 .0136 kcat / Km (mM-1 s-1) 0.0218 0.0126 0.0439 Figure 3-3: Saturation kinetic profile for the oxidation of dopamine ( ), epicatechin (EC) ( ), and epigallocatechin gallate (EGCG) ( ) using Cu2+-A 20 (7 M) at 100 mM HEPES pH 7.4 298K. Tables include kinetic parame ters for dopamine, EC, and EGCG oxidation by Cu2+-A 20.
69 [EGC] mM 0.00.51.01.52.02.53.0 Rate (mM/s) 5.0e-5 1.0e-4 1.5e-4 2.0e-4 2.5e-4 3.0e-4 EGC Cu2+-A 16 Cu2+-A 20 Km (mM) 1.22 0.28 8.91 0.30 Vmax (mM/s) (3.42 0.35) x 10-4 (3.30 0.43) x 10-4 kcat (s-1) .114 .0825 kcat / Km (mM-1 s-1) .0934 .00926 Figure 3-4: oxidation of ep igallocatechin (EGC) by Cu2+-A 20 ( )(4M) and Cu2+A 16 ( )(3M) at 100mM HEPES pH 7.4, 293 K. Table includes kinetic parameters for EGC oxidation by Cu2+-A 16,20.
70 Additionally, the effect of H2O2 on Cu2+-A catalysis was also monitored to obtain both apparent (Kapp) and intrinsic (Km ) affinity constants. (Figure 3-5, 3-6 an d Table 3-2). The Kapp/Km ratio can reveal details on the effect one substrate have on the affinity of other. Ratios above unity indicate one substrate decreases the affinity for the other, while those above unity indicate the opposite. For dopamine, EC, and EGCG, H2O2 seems to have little effect on the binding (clo se to unity). On the contrary, dopamine, EC, and EGCG seem to slightly increase the binding affinity for H2O2. In addition to catechins, green tea is al so an excellent source of ascorbic acid (AsA) and vitamin B6 (B6). Studies have shown both to se rve as excellent antioxidants in addition to other vital roles in the human body. As shown in Figure (3-7,3-8), AsA is an excellent mixed type inhibitor for Cu2+-A 1-16 and Cu2+-A 1-20 toward dopamine oxidation. The most likely explanation is AsA may bind to the metal center and reduce Cu2+ to Cu+ thus preventing O2 from binding. Studi es have shown AsA can increase the stability of GTCs, thus pr oviding benefit by protecting an tioxidants from premature oxidative breakdown. The other component in green tea is one of three derivates of vitamin B6, pyridoxamine. Pyridoxamine is a cr itical component needed by the body, used by some enzymes for the production of neurotransmitters.12 Furthermore, prydoxamine has been shown to inhibit ROS generated in the body.12 As shown in figures (3-9, 3-10) pyridoxamine is a compe titive inhibitor. The competitive inhibition pattern is most likely to be due to pyridoxamine weak affinity for Cu2+.12 This is further supported by the large Ki (in comparison to AsA).
71 [H2O2] mM 0510152025 k cat (s-1) 2e-2 4e-2 6e-2 Figure 3-5: The effect of the concentration of H2O2 on the first-order rate constant kcat toward the A 1-16 oxidation of Dopamine ( ), Epicatechin ( ), and epigallocatechin gallate ( ). [H2O2] mM 051015202530 kcat(s-1) 2.0e-2 4.0e-2 6.0e-2 8.0e-2 1.0e-1 1.2e-1 Figure 3-6: The effect of the concentration of H2O2 on the first-order rate constant kcat toward the A 1-20 oxidation of Dopamine ( ), Epicatechin ( ), and epigallocatechin gallate ( ).
72 Table 3-2: Hanes analysis to compare the apparent (Kapp) and intrinsic (Km) affinity constants of H2O2 on substrate binding and vise versa. Substrate Catalyst Kapp/Km Kapp(H) / Km(H) Dopamine Cu2+-A 16 .924 .228 Dopamine Cu2+-A 20 1.39 1.18 EGCG Cu2+-A 16 1.35 .771 EGCG Cu2+-A 20 1.86 .924 EC Cu2+-A 16 .554 .296 EC Cu2+-A 20 1.43 .824
73 [L-Ascorbic Acid] mM 0.02.0e-34.0e-36.0e-38.0e-31.0e-21.2e-2 Rate (mM/s) 5.0e-7 1.0e-6 1.5e-6 2.0e-6 2.5e-6 3.0e-6 3.5e-6 1/[Dopamine] mM-1 -2 0 2 4 6 8 10 1/Rate mM-1 s 50e+5 10e+6 15e+6 20e+6 25e+6 [Ascorbic Acid] mM A 1-16 Km (mM) Vmax (mM/s) 0.0 0.211 .053 (3.75 0.24) x 10-6 3.0 x 10-4 0.372 .070 (3.25 0.19) x 10-6 1.2 x 10-3 0.560 .178 (2.00 0.23) x 10-6 Ki= 1.37 M Kis= .302 M Figure 3-7: Inhibition of Cu2+-A 1-16 by ascorbic acid (AsA). (Top) AsA titration into fixed [Cu2+-A 1-16] 1 mM Dopamine, 1 mM MB TH. (Bottom) Titrating dopamine at fixed concentra tions of AsA. Table includes effect of [AsA] on kinetic parameters including inhibition constants for mixed-type inhibition. Assays done at pH 7.4 100mM HEPES buffer 293K.
74 [Ascorbic Acid] mM 0.0000.0020.0040.0060.0080.0100.012 Rate (mM/s) 1e-6 2e-6 3e-6 4e-6 5e-6 6e-6 7e-6 1/[Dopamine] mM -1 -20246810 1/Rate mM-1 s 2e+5 4e+5 6e+5 8e+5 1e+6 [Ascorbic Acid] mM A 1-20 Km (mM) Vmax (mM/s) 0.0 0.177 .016 (7.57 0.16) x10-6 5.0 x 10-4 0.248 .026 (7.58 0.21) x 10-6 2.0 x 10-3 0.237 .029 (5.84 0.19) x 10-6 3.0 x 10-3 0.523 .044 (5.61 0.17) x10-6 Ki = 8.59 M Kis = 1.00 M Figure 3-8: Inhibition of Cu2+-A 1-20 by ascorbic acid (AsA). (T op) AsA titration into fixed Cu2+-A 1-20, 1mM Dopamie, 1mM MBTH. (Botto m) Titrating dopamine at fixed concentrations of AsA. Table includes e ffect of [AsA] on kinetic parameters. including inhibition constants for mixed type inhibition. Assays done at pH 7.4 100mM HEPES buffer 293K.
75 [Pyridoxamine] mM 0246 Rate (mM/s) 0 2e-6 4e-6 6e-6 8e-6 [Dopamine] mM 0246810 1/rate (mM-1 s) 5.0e+4 1.0e+5 1.5e+5 2.0e+5 2.5e+5 3.0e+5 [Pyridoxamine] mM A 120 Km (mM) Vmax (mM/s) 0.0 ( ) 0.381 0.063 (2.39 0.14) x10-5 0.5 ( ) 0.590 0.031 (2.74 0.61) x10-5 1.0 ( ) 0.959 0.061 (3.31 0.11) x10-5 Ki= .911 mM Figure 3-9: Inhibition of Cu2+-A 1-20 by Pyridoxamine (B6). (Top) B6 titration into fixed [Cu2+-A 1-20], 1 mM Dopamie, 1mM MBTH. (B ottom) Titrating dopamine at fixed concentrations of B6. Table includes effect of [B6] on kinetic parameters, including inhibition constants for competitive inhibition. Assays done at pH 7.4, 100mM HEPES buffer 293K.
76 [Pyridoxamine] mM 01234567 Rate (mM/s) 0.0 2.0e-6 4.0e-6 6.0e-6 8.0e-6 1.0e-5 1.2e-5 1.4e-5 1/[Dopamine] mM-1 0246810 1/rate (mM-1 s-1) 2.0e+4 4.0e+4 6.0e+4 8.0e+4 1.0e+5 1.2e+5 1.4e+5 1.6e+5 1.8e+5 2.0e+5 2.2e+5 [Pyridoxamine] mM A 1-16 Km (mM) Vmax (mM/s) 0.0 ( ) 0.419 0.020 (3.11 0.05) x 10-5 0.4 ( ) 0.541 0.015 (3.13 0.04) x 10-5 0.8 ( ) 0.746 0.052 (3.37 0.11) x 10-5 Ki= 1.03 mM Figure 3-10: Inhibition of Cu2+-A 1-16 by Pyridoxamine (B6). (Top) B6 titration into fixed Cu2+-A 1-16, 1mM Dopamie, 1mM MBTH (Bottom) Titrating dopamine at fixed concentrations of B6. Table includes effect of [B6] on kinetic parameters. including inhibition constants for competitive inhibition. Assays done at pH 7.4 100mM HEPES buffer, 293K.
77 Quercetin, Fisetin, and Taxifolin There are over 6000 flavonoids which diffe r in both structure and bioactivity.13,14 Studies have been done concerning the bes t structure for the binding of redox-active metal in addition to antioxidant and antiradical activity.13,14 The flavonoids used in this study are structurally similar, differing only by the absence and presence of the enolate or -keto-phenolate. The results show quercetin, fisetin, and taxifolin to be competitive inhibitiors of Cu2+-A oxidation of dopamine. Quercetin contains both the presence of the enolate or -keto-phenolate and shows to be an excellent competitive inhibitor, yielding a Ki (4 M) almost equivalent to the [Cu2+-A ] (Figure 3-11). Fisetin does not contain the -keto-phenolate, but it is near equivelant to querctin in terms of Ki. (Figure 3-12, 3-13). Taxifolin contains the -keto-phenolate but is missing the double bond on the enolate. Taxifolin shows also to be a competitive inhibitor with a Ki 100 times higher then quercetin or fisetin. (Figure 3-15, 3-16) The results, in combination with the fact that catechins are substrates, reveal that the 3-hydroxy on the C-ri ng is required for inhibition. Furthermore the presence of th e enolate allows for a better affinity for Cu2+. To further distinguish between the -keto-phenolate and enolat e, fisetin was used in conjunction with Ca2+. The Ca2+ titration in figure (3-14) reveal that Ca2+ attenuate the inhibition of fisetin oxidation of dopamine by Cu2+-A 16. This result concludes that Ca2+ is binding in the same place as Cu2+ the enolate. The general mechanism for AD is believed to involve metal-centers ROS. This study proposes the use of green tea components as suicide substrates and antioxidants for a possible way to slow the oxidation of neurotransmitters. In addition, citrus flavonoids quecetin, fisetin, taxifolin have shown to be excellent inhibitors of both Cu2+-A 16 and
78 Cu2+-A 20. The Ca2+ binding properties allude to ce rtain flavonoids to serve as Ca2+ sponge, potentially reducing the influx of Ca2+ seen in AD. The activity and inhibition of both the Cu2+-A 16 and Cu2+-A 20 conclude that the 4 amino acid differences make no significant contribu tion to activity.
79 [Quercetin] mM 0.0000.0050.0100.01 50.0200.0250.030 Rate (mM/s) 2.0e-6 4.0e-6 6.0e-6 8.0e-6 1.0e-5 1.2e-5 1.4e-5 1.6e-5 1.8e-5 1/[Dopamine] mM -50510152025 1/Rate (mM-1 s) 0.0 2.0e+5 4.0e+5 6.0e+5 8.0e+5 1.0e+6 1.2e+6 [Quercetin] mM A 16 Km (mM) Vmax (mM/s) 0 0.467 0.053 (3.26 0.15) x 10-5 2.0 x 10-3 0.699 0.052 (3.59 0.13) x10-5 4.0 x 10-3 0.962 0.082 (3.92 0.18) x 10-5 6.0 x 10-3 1.31 0.16 (4.39 0.29) x 10-5 8.0 x 10-3 1.57 0.12 (4.32 0.19) x 10-5 Ki= .004 mM Figure 3-11: Quercetin inhibition of Cu2+-A 1-16 oxidation of dopamine. (Top) Quercetin titration into fixed [Cu2+-A 1-16], [Dopamine]. [MBTH]. (Bottom) Titrating dopamine at fixed concentrations of quercetin Table includes effect of [Quercetin] on kinetic parameters including inhibition constants for compet itive inhibition. Assays done at pH 7.0 100mM HEPES buffer 293K. ( ), .002( ), .004 ( ), .006( ), .008mM ( ) of inhibitor
80 [ Fisetin ] mM 0.000.050.100.18.104.22.1680.35 Rate (mM/s) 2e-6 4e-6 6e-6 8e-6 1e-5 1/[Dopamine] mM -1 -20246810 1/rate mM-1 s 0 1e+6 2e+6 3e+6 4e+6 [Fisetin] mM A 1-20 Km (mM) Vmax (mM/s) 0 0.307 0.039 (9.22 0.37) x10-6 1.25 x 10-2 0.756 0.148 (8.12 0.75) x10-6 2.50 x 10-2 1.73 0.35 (9.83 0.13) x 10-6 5.00 x 10-2 2.03 0.78 (7.40 0.19) x10-6 Ki= .009 mM Figure 3-12: Fisetin inhibition of Cu2+-A 1-20 oxidation of dopamine. (Top) Fisetin titration into fixed [Cu2+-A 1-16], [Dopamine]. [MBTH]. (Bottom) Titrating dopamine at fixed concentrati ons of fisetin. Table incl udes effect of [fisetin] on kinetic parameters including inhibition constants for competitive inhibition. Assa y s done at p H 7.4, 0 mM ( ) .0125 ( ) .025 ( ) .05 ( ) ,of inhibitor.
81 [Fisetin] mM 0.000.050.100.22.214.171.1240.35 Rate (mM/s) 2e-6 4e-6 6e-6 8e-6 1/[dopamine] mM-1 -20246810 1/rate mM-1 s 0.0 5.0e+5 1.0e+6 1.5e+6 2.0e+6 2.5e+6 3.0e+6 3.5e+6 [Fisetin] mM A 1-16 Km (mM) Vmax (mM/s) 0.00 0.271 0.040 (8.71 0.39) x 10-6 1.25 x 10-2 0.830 0.176 (7.97 0.83) x 10-6 2.50 x 10-2 1.31 0.34 (8.31 1.24) x 10-6 5.00 x 10-2 2.32 0.69 (7.59 1.55) x10-6 Ki= .007 mM Figure3-13: Fisetin inhibition of Cu2+-A 1-16 oxidation of dopamine. (Top) Fistein titration into fixed [Cu2+-A 1-16], [Dopamine]. [MBTH]. (Bottom) Titrating dopamine at fixed concentrations of fistein. Table includes eff ect of [fistein] on kinetic parameters including inhibition constants for competitive inhibition. Assays done at pH 7.4, 0 mM ( ), .0125( ), .025 ( ), .05( ),of inhibitor.
82 [CaCl2] mM 05101520253035 Rate (mM/s) 3.0e-6 3.5e-6 4.0e-6 4.5e-6 5.0e-6 5.5e-6 6.0e-6 6.5e-6 [Dopamine] mM 0.00.51.01.52.02.5 Rate (mM/s) 2e-6 4e-6 6e-6 8e-6 1e-5 Fisetin Ca2+ A 1-16 Fistein 0 mM Fistein .0 25 mM Fistein .025 mM Ca2+ Km (mM) 0.271 0.040 1.310 0.337 0.806 0.088 Vmax (mM/s) (8.71 0.39) x 10-6 (8.32 1.24) x 10-6 (8.73 0.41) x 10-6 kcat s-1 0.003 0.003 0.003 kcat /Km (mM-1 s-1) 0.011 0.002 0.004 Figure 3-14: Ca2+ effect on Fistei n inhibition of Cu2+-A 1-16 oxidation of dopamine. (Top) Ca2+ titration in fixed [Cu2+-A 1-16], [Dopamine], and [Fisetin]. (Bottom) Dopamine titrations without fisetin ( ), with fisetin ( ), and with fisetin + Ca2+ ([Ca2+] = 30 mM)( ). Table includes kinetic parameters.
83 [Taxifolin] mM 000.20.40.60.81.01.21.41.6 Rate (mM/s) 1e-6 2e-6 3e-6 4e-6 5e-6 6e-6 1/[Dopamine] mM-1 024681 0 1/Rate (mM-1 s) 0 1e+5 2e+5 3e+5 4e+5 5e+5 6e+5 7e+5 [Taxifolin] mM A 1-16 Km (mM) Vmax (mM/s) 0.00 0.271 0.040 (8.71 0.39) x 10-6 0.16 0.392 0.075 (8.38 0.57) x 10-6 0.32 0.670 0.149 (9.66 0.97) x 10-6 0.64 1.23 0.282 (1.14 0.15) x10-5 Ki = 0.216 mM Figure3-15: Taxifolin inhibition of Cu2+-A 1-16 oxidation of dopamine. (Top) Taxifolin titration into fixed [Cu2+-A 1-16], [Dopamine]. [MBTH]. (Bottom) Titrating dopamine at fixed concentrations of taxifolin. Tabl e includes effect of [Taxifolin] on kinetic parameters including inhibition constants for competitive inhibition. Assays done at pH 7.4, 0 ( ), .16 ( ), .32 ( ), .64 mM( ),of inhibitor.
84 [Taxifolin] mM 0.00.20.40.60.81.01.21.41.6 Rate (mM/s) 1e-6 2e-6 3e-6 4e-6 5e-6 6e-6 7e-6 8e-6 9e-6 1/[dopamine] mM -1 -20246810 1/Rate mM-1 s 0 1e+5 2e+5 3e+5 4e+5 5e+5 6e+5 [Taxifolin] mM A 1-20 Km (mM) Vmax (mM/s) 0.00 0.290 .0585 (1.19 0.08) x 10-5 0.16 0.331 .0663 (1.15 0.08) x 10-5 0.32 0.539 .102 (1.10 0.09) x 10-5 0.64 0.729 .150 (1.12 0.11) x 10-5 Ki= 0.372 mM Figure3-16: Taxifolin inhibition of Cu2+-A 1-20 oxidation of dopamine. (Top) Taxifolin titration into fixed [Cu2+-A 1-20], [Dopamine]. [MBTH]. (Bottom) Titrating dopamine at fixed concentrations of taxifolin. Tabl e includes effect of [Taxifolin] on kinetic parameters including inhibition constants fo r competitive inhibition. Assays done at pH 7.4, 0 ( ), .16 ( ), .32 ( ), .64 mM( ),of inhibitor.
85 Closing Remarks With plaques composed of A in addition to redox active metal, the results indicate their combination can result in oxid ative damage. As shown, different forms of metallo-A can oxidative neurotransmitters which ma y be a cause or effect of AD. This thesis focuses on the possible use of natural an tioxidants to slow or inhibit this oxidative damage. Flavonoids have been studied ex tensively and are considered to provide therapeutic effect for numerous diseases. He re, the GTCs were shown to be oxidized and can potentially serve as suicide substrates. In addition vitamins AsA and B6 were shown to inhibit the metallo-A redox chemistry, possibly by reducing or chelating the metal. This study extents into the debate over th e best flavonoid, by examining the properties of several common moieties. The GTCs, quercetin, fisetin, and taxifolin all vary specific functional groups and through inhibition of metallo-A can determine thoughts of most benefit. The results indicate that the enolat e is the most important in terms of metal chelation. The absence of the -keto-phenolate seems to have no effect on the inhibition, while the opposite it true for the 2-3 alkene.
86 Reference 1) Gaggelli, E,. Kozlowski, H,. Valensin D,. and Valensin G. (2006) Copper homeostasis and neurodegenerative disorder s (Alzheimer's, pri on, and Parkinson's diseases and amyotrophic lateral sc lerosis). Chem. Rev., 106, 1994-2044. 2) Huang, X., Atwood, S, C., Hartshorn, A, M., Multhaup, G., Goldstein, E, L., Scarpa, C, R., Cuajungco. P, M., Gray, N, D., Lim, J., Moir, D, R., Tanzi, E, R. and Bush, I, A. (1999) The A Peptide of Alzheimer's Disease Directly Produces Hydrogen Peroxide through Metal Ion Reduction. Biochemistry, 38, 7609 -7616 3) Xie, C, W. (2004) Calcium-regulated signa ling pathways: role in amyloid beta-induced synaptic dysfunction. Neuromolecular Med., 6, 53-64. 4) Huang, X., Moir, D, R., Tanzi, E, R., Bush, I., A., and Rogers, T, R. (2004) RedoxActive Metals, Oxidative Stress, and Alzheimer's Disease Pathology. Ann. N.Y. Acad. Sci. 1012, 153163. 5) Mekmouche, Y., Coppel, Y., Hochgrafe, K., Guilloreau, L., Talmard, C., Mazarguil, H. and Faller, P. (2005) Characterization of the ZnII binding to the peptide amyloidbeta1-16 linked to Alzheimer's dis ease. Chembiochem., 6, 1663-71. 6) Gnanakaran, S., Nussinov, R. and Garcia, A. E. (2006) Atomic-Level Description of Amyloid -Dimer Formation. J. Am. Chem. Soc. 128, 2158-2159. 7) Da Silva, GFZ., Tay, WM., and Ming, L. (2005) Catechol Oxidase-like Oxidation Chemistry of the 120 and 116 Fragment s of Alzheimer's Disease-related -Amyloid Peptide J. Bio. Chem., 280, 16601-16609. 8) Mattson, M. (2000) Apoptos is in Neurodegenerative Disord ers. Mol. Cell. Bio., 1, 120-129.
879) Casetta, I., Govni, V., and Granieri E. (2005) Oxidative stress, antioxidants and neurodegenerative diseases. Curr. Pharm. Des., 11, 2033-52. 10) Choi, Y, T., Jung, C, H., Lee, S, R., Bae, J, H., Baek, W, K., Suh, M, H., Park, J., Park, C, W. and Suh, S, I. (2001) The green tea polyphenol (-)-epigallocatechin gallate attenuates beta-amyloid-induced neurotoxicity in cultured hippocampa l neurons. Life Sci. 70, 603-614. 11) Weinreb, O., Mandel, S., Amit, T., and Youdim, M. (2004) Neurological mechanisms of green tea polyphenols in Alzheimers and Parkinsons diseases. J. of Nutrition. Biochem. 15, 506-516. 12) Voziyan, A, P. and Hudson, G, B. (2005) Pyridoxamine: The Many Virtues of a Maillard Reaction Inhibitor. Ann. N.Y. Acad. Sci., 1043, 807816. 13) Matsuda, H., Wang, T., Managi, H. and Yoshikawa, M. (2003) Structural requirements of flavonoids fo r inhibition of protein glyc ation and radical scavenging activities. Bioorganic and Medicinal Chem., 11, 5317-5323. 14) Thompson, M. and Williams, C. R. (1976) Antioxidant Potential of Ecklonia cavaon Reactive Oxygen Species Scavenging, Meta l Chelating, Reducing Power and Lipid Peroxidation Inhibition. Anal ytica Chimica Acta, 85, 375-381.
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Juneja, Kashmir Singh.
Tyrosinase-like activity of several Alzheimer's disease related and model peptides and their inhibition by natural antioxidants
h [electronic resource] /
by Kashmir Singh Juneja.
[Tampa, Fla] :
b University of South Florida,
ABSTRACT: Neurodegenerative diseases are associated with loss of neurons ultimately leading to a decline in brain function. Alzheimer's disease (AD) is considered one of the most common neurodegenerative disorders that affects 16 million people worldwide. The cause of the disease remains unknown, although significant evidence proposes the amyloid Beta-peptide (A-Beta) as a potential culprit. The binding of Cu2+ by the soluble fragments of A-Beta have shown to form Type-3 copper centers and catalyze the oxidation of catechol-containing neurotransmitters. Furthermore, the use of flavonoids as antioxidants to slow or inhibit the neurotransmitter oxidation has suggested further health benefits with their consumption. A structure-function correlation is also made between the flavonoids and their reactively with Cu2+-A-Beta. Mechanistic insight into the binding of catechol and dioxygen within the tyrosinase-like mechanism are made using a metallopeptide modeling the active site of the metzinicins.
Thesis (M.S.)--University of South Florida, 2006.
Includes bibliographical references.
Text (Electronic thesis) in PDF format.
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Mode of access: World Wide Web.
Title from PDF of title page.
Document formatted into pages; contains 87 pages.
Adviser: Li-June Ming, Ph.D.
t USF Electronic Theses and Dissertations.