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Biochemical Investigations of L-Methionine Gamma-Lyase 1 from Trichomonas Vaginalis

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Biochemical Investigations of L-Methionine Gamma-Lyase 1 from Trichomonas Vaginalis Biochemical Investigations of L-Methionine gamma-lyase 1 from Trichomonas vaginalis by Ignace Adolfo Moya A thesis presented to the University of Waterloo in fulfillment of the thesis requirement for the degree of Doctor of Philosophy in Chemistry Waterloo, Ontario, Canada, 2011 © Ignace Adolfo Moya 2011 AUTHOR'S DECLARATION I hereby declare that I am the sole author of this thesis. This is a true copy of the thesis, including any required final revisions, as accepted by my examiners. I understand that my thesis may be made electronically available to the public. ii ABSTRACT The enzyme L-methionine γ-lyase (MGL) utilizes a pyridoxal-5’-phosphate-cofactor in order to convert L-methionine to α-ketobutyrate, ammonia and methyl mercaptan. MGL is proposed to be a potential drug target since it is expressed in the human pathogens, Trichomonas vaginalis and Entamoeba histolytica, but not in humans. There is currently a need to find alternative drug targets for these pathogens, because the misuse and overuse of the currently prescribed drugs of choice, metronidazole and tinidazole, have lead to drug resistance. The overall goal of this thesis was to examine the chemistry of MGL 1 from T. vaginalis (TvMGL1) by probing the active site by site-directed mutagenesis and with fluorinated methionine analogs. The mutation of the active site residue Cys113 to Ser led to a 5-fold decrease in turnover rate for L-methionine relative to the wild-type enzyme. The results suggest that the active site C113 residue plays an important role in catalysis and is consistent with literature reports for MGL homologs from Pseudomonas putida and E. histolytica. Probing the active site of TvMGL1 with the fluorinated methionine analogs, L- difluoromethionine (DFM) and L-trifluoromethionine (TFM), were found to increase the turnover rate of the enzyme with an increase in fluorine substitution. The results suggest that the bulky fluorine atoms do not interfere with the Michaelis-Menten kinetics of the enzyme, and the γ-elimination step is rate determining. iii The second goal of this thesis was to identify the reactive intermediates generated by the processing of TFM and the uninvestigated DFM by TvMGL1, and to investigate the theoretical and experimental chemistry and biochemistry of these fluorinated groups (CF3S- and CF2HS-). The reactivity of the intermediates, generated from the processing of DFM by TvMGL1 was correlated to the cytotoxicity observed in model organisms expressing TvMGL1, and consistent with the hypothesis that the intermediates will result in the thioformylation of primary amines. The results suggest that cytotoxicity requires thioacylation of a single primary amine, while sequential cross-linking of primary amines is not an absolute requirement. The relationship between the chemical structure of the reactive intermediates produced from the enzymatic processing of these analogs and their cellular toxicity is discussed. Attempts at the synthesis of 3,3-difluoro-O-methyl-L-homoserine were undertaken in order to examine the catalytic mechanism of TvMGL1, since the compound is expected to inhibit the enzyme. To ensure that the oxo moiety does not impede the chemistry of the enzyme, the analog, O-methyl-L-homoserine was examined as a potential substrate for TvMGL1. Several synthetic routes to 3,3-difluoro-O-methyl-L-homoserine were examined; however, attempts to fluorinate the β-carbon atom of the starting material were unsuccessful. iv ACKNOWLEDGEMENTS I would like to thank my supervisor, Dr. John Honek for his help and support with the project, and improving my understanding of the field. I enjoyed the insightful discussions we had over a cup of coffee and during lunch about medicinal chemistry. My committee members, Drs. E. Meiering, K. Ma, A. Schwan for their time and critique of my work. Dr. R. Smith (mass spectrometry), C. Myers (mass spectrometry) for obtaining the ESI-MS data. J. Venne (NMR) for running some of my samples, training and for her helpful suggestions. Dr. S. Taylor, Dr. M. Chong and J. Su for their suggestions in trouble shooting the chemical reactions. To our collaborators, Drs. G. Coombs and G. Westrop (University of Strathclyde, Glasgow) for testing the compound on the parasite and critiquing the published manuscript. I appreciate all of my friends and colleges for their support during my time in Waterloo: T. Trecroce, A. Cavanagh, O. Zhang, J. DaCosta, P. Hang, S. Zhu, V. Azhikannickal, J. Wang, J. Dubrick,Q. Bahtti, M.Grewal, K. Kosar, J. Warren, J. Stewart, K. Mullings, R. Zahoruk, U. Suttisansanee, J. Harris, Dr. E. Daub, Dr. Z. Su, J. Verhoek, M. Vanderkooy, D. Fuhr, R. Clark and E. Ohm. Special thanks to my former supervisor Dr. Y. Luo (University of Saskatchewan, Saskatoon, SK) and Dr. C. Phenix (TBRRI, Thunder Bay, ON) for their help with editing of my manuscript, suggestions, advice, laboratory training, and past and current mentoring. v DEDICATIONS Dedicated to my parents and sister for their support in my endeavours. And the early bird that was already in bed while I worked away until the late hours of the night. vi TABLE OF CONTENTS AUTHOR'S DECLARATION ................................................................................................. ii ABSTRACT ............................................................................................................................. iii ACKNOWLEDGEMENTS ...................................................................................................... v DEDICATIONS ....................................................................................................................... vi TABLE OF CONTENTS ........................................................................................................ vii LIST OF FIGURES ................................................................................................................. xi LIST OF TABLES ................................................................................................................. xiv LIST OF ABBREVIATIONS ................................................................................................. xv CHAPTER 1 BACKGROUND ................................................................................................ 1 1.1. Authors’ Contributions ................................................................................................... 1 1.2. Introduction .................................................................................................................... 1 1.3. Epidemiology and Treatment for Parasitic Infections.................................................... 3 1.4. Potential Drug Target for Parasitic Infections................................................................ 8 1.5. Superfamilies of PLP-dependent Enzymes .................................................................. 12 1.6. Reactions Catalyzed by L-Methionine γ-lyase............................................................. 19 1.7. Methionine-dependent Tumors .................................................................................... 22 1.8. Cysteine Biosynthesis Pathway .................................................................................... 24 1.8.1. An Overview of Sulfur Biochemistry in Living Organisms ................................. 24 1.8.2. Methionine Catabolism ......................................................................................... 27 1.8.3. Sulfide Biosynthetic Pathway and de novo Cysteine Biosynthesis ....................... 29 1.9. Purpose of Study .......................................................................................................... 31 CHAPTER 2 ROLE OF THE ACTIVE SITE RESIDUE C113 IN TRICHOMONAS VAGINALIS L-METHIONINE γ-LYASE 1 ........................................................................... 32 2.1. Authors’ Contribution .................................................................................................. 32 2.2. Introduction .................................................................................................................. 32 2.3. Studies on the Functional Role of the Cysteine Residue ............................................. 33 2.4. Results and Discussion ................................................................................................. 37 vii 2.5. Conclusion and Future Work ....................................................................................... 39 2.6. Materials and Methods ................................................................................................. 40 2.6.1. General Experimental ............................................................................................ 40 2.6.2. TvMGL1 C113 Mutants ........................................................................................ 40 2.6.3. Agarose Gel Electrophoresis ................................................................................. 42 2.6.4. Cell Culture and Expression of Enzyme ............................................................... 42 2.6.5. Cell Lysis and Protein Purification ........................................................................ 43 2.6.6. SDS-Polyacrylamide Gel Electrophoresis ............................................................. 45 2.6.7. MBTH Assay Method for Detecting α-Ketobutyrate ............................................ 46 CHAPTER 3 MECHANISTIC STUDIES ON THE ENZYMATIC PROCESSING OF FLUORINATED METHIONINE ANALOGS BY TRICHOMONAS VAGINALIS L- METHIONINE γ-LYASE 1 ..................................................................................................
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