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Saccharomyces Cerevisiae Aspartate Kinase Mechanism and Inhibition

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Saccharomyces Cerevisiae Aspartate Kinase Mechanism and Inhibition In compliance with the Canadian Privacy Legislation some supporting forms may have been removed from this dissertation. While these forms may be included in the document page count, their removal does not represent any loss of content from the dissertation. Ph.D. Thesis - D. Bareich McMaster University - Department of Biochemistry FUNGAL ASPARTATE KINASE MECHANISM AND INHIBITION By DAVID C. BAREICH, B.Sc. A Thesis Submitted to the School of Graduate Studies in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy McMaster University © Copyright by David C. Bareich, June 2003 1 Ph.D. Thesis - D. Bareich McMaster University - Department of Biochemistry FUNGAL ASPARTATE KINASE MECHANISM AND INHIBITION Ph.D. Thesis - D. Bareich McMaster University - Department of Biochemistry DOCTOR OF PHILOSOPHY (2003) McMaster University (Biochemistry) Hamilton, Ontario TITLE: Saccharomyces cerevisiae aspartate kinase mechanism and inhibition AUTHOR: David Christopher Bareich B.Sc. (University of Waterloo) SUPERVISOR: Professor Gerard D. Wright NUMBER OF PAGES: xix, 181 11 Ph.D. Thesis - D. Bareich McMaster University - Department of Biochemistry ABSTRACT Aspartate kinase (AK) from Saccharomyces cerevisiae (AKsc) catalyzes the first step in the aspartate pathway responsible for biosynthesis of L-threonine, L-isoleucine, and L-methionine in fungi. Little was known about amino acids important for AKsc substrate binding and catalysis. Hypotheses about important amino acids were tested using site directed mutagenesis to substitute these amino acids with others having different properties. Steady state kinetic parameters and pH titrations of the variant enzymes showed AKsc-K18 and H292 to be important for binding and catalysis. Little was known about how the S. cerevisiae aspartate pathway kinases, AKsc and homoserine kinase (HSKsc), catalyze the transfer of the y-phosphate from adenosine triphosphate (ATP) to L-aspartate or L-homoserine, respectively. Two transfer paths are possible as are a range of mechanisms, with two extremes. Both kinases were shown to directly transfer the y-phosphate from ATP to the amino acid. Experimental evidence was consistent with an associative mechanism of phosphoryl transfer but did not rule out a dissociative mechanism. AKSc and HSKsc are good targets for inhibitors because they catalyze phosphoryl transfers between very similar substrates. An attempt was made to rationally design inhibitors to these kinases by linking the substrates with a variable length linker to create bisubstrate compounds. This strategy failed inhibit AKsc and HSKsc, however, one of the bisubstrate compounds was a good inhibitor of AKIII from Escherichia coli. 111 Ph.D. Thesis - D. Bareich McMaster University - Department of Biochemistry Inhibition of any of the enzymes in the aspartate pathway would lead to reduced production of amino acids. A pathway assay was optimized to allow screening of chemical libraries in the hope of identifying inhibitors to the first four pathway enzymes. A high throughput screen of 1,000 compounds identified two compounds capable of inhibiting the assay, one of which was the best inhibitor identified for AKsc, IV Ph.D. Thesis - D. Bareich McMaster University - Department of Biochemistry ACKNOWLEDGEMENTS I would like to acknowledge past members of the Wright lab for passing their experience on to me, setting the standard of science and establishing an excellent reputation for the lab. I would like to acknowledge current members of the lab for insightful discussions and particularly Ishac Nazi for cloning several of the enzymes I used in my work and Dr. Kalinka Koteva for synthesis of the bisubstrate inhibitors. I would like to thank members of my Graduate Committee, which included Dr. David Andrews, Dr. Gerhard Gerber, and Dr. Paul Harrison, for their helpful suggestions and encouraging excellence. I extend special thanks to my supervisor, Dr. Gerry Wright, for his mentoring, guidance, encouragement, advice, and teaching me much about the science of enzymes and inhibitors. I would like to thank old friends and friends I've made during my studies for their friendship. I would also like to thank the family I happily married into, for their generous love and encouragement. Thanks goes out to my close family for being there for me. I have no doubt that I wouldn't have written this without you. Lastly, to my wife, for being there everyday with love, humour, and companionship. v Ph.D. Thesis - D. Bareich McMaster University - Department of Biochemistry For my mother VI Ph.D. Thesis - D. Bareich McMaster University - Department of Biochemistry TABLE OF CONTENTS Abstract III Acknowledgements V Table of Contents Vll List of Tables Xlll List of Figures XIV List of Schemes XVI List of Abbreviations XVll Chapter 1. Introduction 1 1.1. The Need for Antifungal Agents 2 1.2. Current Antifungal Agents 7 1.3. Mechanisms of Antifungal Resistance 15 1.4. Fungicides and Resistance to Fungicides .20 1.5. The Aspartate Pathway: A New Antifungal Target 29 1.6. The Enzymes of the Aspartate Pathway 37 1.6.1. Aspartate Kinase 37 1.6.2. Aspartate Semialdehyde Dehydrogenase 41 1.6.3. Homoserine Dehydrogenase 43 1.6.4. Homoserine O-Acetyl Transferase 45 1.6.5. Homoserine Kinase 47 1.6.6. Threonine Synthase 48 1.7. General Mechanisms of Kinases 49 Vll Ph.D. Thesis - D. Bareich McMaster University - Department of Biochemistry 1.8. Exploring the Aspartate Pathway: Building on What is Known 54 1.9. References 55 Chapter 2. Identification of Amino Acids Important to Catalysis in Saccharomyces cerevisiae Aspartate Kinase 77 2.1. Preface 78 2.2. Abstract 79 2.3. Introduction 80 2.4. Results and Discussion 82 2.4.1. Expression and Purification of AKsc 82 2.4.2. Analysis of AKsc Engineered Variants 82 2.4.2.1. Signature Motif for AKs 82 2.4.2.2. AKsc-Histidine 497 84 2.4.2.3. Amino Acid Similarity Between AKs and Homoserine Kinases 84 2.4.2.4. Amino Acid Similarity Between AKsc and N-Acetylglutamate Kinase 89 2.4.3. Effect of pH on the Steady State Kinetic Parameters of AKsc and K18A, H292A, and H497A Variants 90 2.5. Materials and Methods 92 Vll1 Ph.D. Thesis - D. Bareich McMaster University - Department of Biochemistry 2.5.1. Cloning of AKsc 92 2.5.2. Mutagenesis ofpET28+AKsc 92 2.5.3. Wild-type and Mutant AKsc Expression and Purification 93 2.5.4. AK Assay and Determination of Steady State Kinetic Parameters 94 2.5.5. L-Thr ICso Determinations 95 2.5.6. pH Experiments 96 2.6. Conclusion 97 2.7. Acknowledgements 98 2.8. References 98 Chapter 3. Small Molecule Functional Discrimination of the Kinases Required for the Microbial Synthesis of Threonine and Isoleucine 111 3.1. Preface 112 3.2. Abstract 113 3.3. Introduction 114 3.4. Results and Discussion 116 3.4.1. Steady State Characterization of the Aspartate Pathway Kinases 116 3.4.2. AK and HSK Have Similar Mechanisms ofPhosp~oryl Transfer 117 IX Ph.D. Thesis - D. Bareich McMaster University - Department of Biochemistry 3.4.3. Small Molecule Inhibitors of AKsc, AKIIIEc , and HSKsp 119 3.4.4. Bisubstrate Compound Inhibition of the Aspartate Pathway Kinases 120 3.5. Conclusions 122 3.6. Acknowledgements 123 3.7. Experimental 123 3.7.1. AKsc cloning, expression, and purification 124 3.7.2. AKIIIEc cloning, expression, and purification 123 3.7.3. Schizosaccharomyces pombe HSK cloning, expression, and purification 126 3.7.4. Enzyme Assays 127 3.7.5. Product Inhibition Studies for AKsc 128 3.7.6. Data Fitting 128 3.7.7. Detection ofa Stable Phospho enzyme Intermediate 130 3.7.8. ATPase Assay 131 3.7.9. Preparation of compounds 1 - 4 131 3.7.10. Preparation ofbisubstrate compounds 5 - 8 from 1- 4 133 3.8. References 134 x Ph.D. Thesis - D. Bareich McMaster University - Department of Biochemistry Chapter 4. Simultaneous In Vitro Assay of the First Four Enzymes in the Fungal Aspartate Pathway Identifies a New Class of Aspartate Kinase Inhibitor 147 4.1. Preface 148 4.2. Summary 149 4.3. Introduction 150 4.4. Results and Discussion 152 4.5. Significance 156 4.6. Experimental Procedures 157 4.6.1. Cloning, Expression, and Purification of Fungal Aspartate Pathway Enzymes. 157 4.6.2. ADP Production Assay 159 4.6.3. L-Threonine ICso Determinations 160 4.6.4. NADPH Oxidation Assay 161 4.6.5. Assay for CoASH Production 161 4.6.6. Optimization of the Pathway Assay 162 4.6.7. Enzyme Screening 163 4.6.8. Data Analysis and Characterization of Inhibitory Compounds 163 4.7. Acknowledgements 165 4.8. References 165 Chapter 5. Discussion and Future Work 175 Xl Ph.D. Thesis - D. Bareich McMaster University - Department of Biochemistry 5.1 Contributions Towards Understanding How Aspartate Kinase Catalyzes Phosphoryl Transfer 176 5.2 Inhibition of Aspartate Kinase 177 5.2.1 Rational Design of Inhibitors to Aspartate Kinase 178 5.2.2 Screening a Chemical Library for Inhibitors to the First Four Enzymes ofthe Aspartate Pathway 179 XlI Ph.D. Thesis - D. Bareich McMaster University - Department of Biochemistry LIST OF TABLES 1.1. Clinically important antifungals. 11 1.2. Agriculturally important fungicides. 26 2.1. Purification of AKsc from 4 L of E. coli BL2l(DE3)/ pET28+AKsc. 102 2.2. Steady state kinetics for AKSc and engineered variants. 103 2.3. L-Thr Ki values and mechanism of inhibition for AKSc and AKsc-E279A. 104 2.4. Summary of pH effects on the steady state kinetic parameters of AKsc and select engineered variants 105 2.5. PCR primers used for site directed mutagenesis of AKsc. 106 3.1. Steady state kinetic parameters for AKSc, AKIIIEc, HSKsp. 139 3.2. IC50 values for inhibitors of AKsc, AKIIIEc, and HSKsp. 140 3.3. IC50 values for the bisubstrate compounds. 141 3.4.
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