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Proquest Dissertations BACTERIAL PHOSPHATIDYLINOSITOL-SPECIFIC PHOPHOLIPASE C: INSIGHTS INTO ENZYMATIC MECHANISM THROUGH NMR, PROTEIN ENGINEERING, AND LINEAR FREE ENERGY RELATIONSHIPS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University By Alexander V. Kravchuk, M.S. ***** The Ohio State University 1999 Dissertation Committee: Approved by Professor Ming-Daw Tsai, Adviser Professor Lawrence Berliner Professor Dehua Pei Adviser Department of Chemistry IUVfl Number: 9951683 U l V d L l " UMI Microform 9951683 Copyright 2000 by Bell & Howell Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. Bell & Howell Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, MI 48106-1346 ABSTRACT Structure, function, and mechanism of phosphatidylinositol specific phospholipase C (PI-PLC) from Bacillus thuringiensis have been studied by multidimensional nuclear magnetic resonance (NMR) spectroscopy, protein engineering, and linear free energy relationships (LFER). Determination of the pKaS of the histidine side chains in the wild type (WT) enzyme and two active site mutants has conclusively established a complex nature of the PI-PLC’s pH-rate profile, where decrease of the activity at the extreme pHs cannot be explained by simple protonation of a general base or deprotonation of a general acid. Most likely, pH-rate profiles of PI-PLC are governed by elaborate acid-base equilibria in the enzyme’s active site, effecting its extensive hydrogen bond network. A single active site mutation, arginine-69 to aspartate, has created a metal binding site with sub-millimolar affinity for both magnesium and calcium ions. Metal ions have partially restored mutant’s activity, but even fully activated R69D falls far below the WT’s level. Further examination of the created metal binding site has revealed that it is composed of aspartate-33, aspartate-67, aspartate-69, and glutamate-117. D33N/R69D double mutant favors magnesium over calcium ions, albeit the discrimination comes at the expense of slightly lower activity. Sigmoidal nature of the rate vs. metal ion concentration plots for the double mutant suggests that the second mutation has created a molecular switch in the active site, which is turned on by the addition of a metal ion. Site-directed chemical modification has been used to elaborate function of arginine-69 in the enzyme catalysis. Consequences of the structure of the side chain at that position on enzyme’s activity, non-bridging thio-effects, and stereoselectivity have been systematically examined. The results have established that bi-dentate functional group in the place of Arg69 is essential for the enzyme’s activity. The proposal that arginine-69 interacts with both pro-S oxygen of the phosphate group and 2-OH of inositol (incoming nucleophile in the reaction) seems to be the most consistent with the available functional and structural data. LFER studies have examined effects of the pKa of the leaving group on the rates of both enzymatic and non-enzymatic reactions. Analysis of the Bronsted coefficients established that both enzymatic and imidazole-catalyzed reactions proceed through essentially identical, slightly dissociative, transition states. Additionally, it has been shown that replacement of a non-bridging phosphate oxygen by sulfur does not alter reactivity of the inositol phosphate diesters. Ill ACKNOWLEDGMENTS I would like to thank my adviser, Ming-Daw Tsai, for his support, both intellectual and financial, his encouragement and patience throughout of ups and downs of my graduate career. Most of this work would be impossible without advice and intellectual input from professor Karol Bruzik from University of Illinois at Chicago. His lab has also supplied numerous substrate analogs used in this work. 1 have also learned a lot of synthetic organic chemistry during my ten week sabbatical in Prof. Bruzik's lab in summer of 1998 with the help and under the supervision by Robert Kubiak. I am grateful to Dr. In Ja Beoyn and Dr. Charles Cottrell from OSU Campus Chemical Instrument Center for teaching me high-field NMR of proteins and for their patience in solving my numerous problems and answering even more numerous questions related to my NMR experiments. Dr. Karl Vermillion provided an excellent technical support at the NMR laboratory of OSU Department of Chemistry. I wish to thank Hua Liao for helpful discussions and valuable help with various aspects of the PI-PLC project. Li Zhao provided indispensable assistance in site-directed mutagenesis and protein purifications. 1 am indebted to Dr. Suzette Riddle for her help and instructions in the beginning of my graduate research. IV VITA February 3, 1967 ..................................... Bom - Slavuta, Ukraine 1989..........................................................M.S., Moscow State University, Russia 1989 - 1993............................................. Researcher, Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Russia 1993 - present..........................................Graduate Teaching and Research Associate, The Ohio State University PUBLICATIONS 1. Hondal, R.J., Zhao, Z., Kravchuk. A V.. Liao, H., Riddle, S R., Bruzik, K.S. and Tsai, M.-D. (1999) “Mechanism of phosphatidylinosito 1-specific phospholipase C revealed by protein engineering and phosphorothioate analogs of phosphatidylinositol”. ACS Symp. Sen, 718, 109-120. 2. Hondal, R. J.; Zhao, Z.; Kravchuk. A. V.: Liao, H.; Riddle, S. R.; Yue, X.; Bruzik, K. S.; Tsai, M.-D. “Mechanism of phosphatidylinosito 1-specific phospholipase C: a unified view of the mechanism of catalysis”. Biochemistry (1998), 37(13), 4568-4580. 3. Hondal, R. J.; Zhao, Z.; Riddle, S. R.; Kravchuk. A. V : Liao, H.; Bruzik, K. S.; Tsai, M.-D. “Phosphatidylinositol-specific phospholipase C. 3. Elucidation of the catalytic mechanism and comparison with ribonuclease A.” J. Am. Chem. Soc. (1997), 119(41), 9933-9934. 4. Hondal, R. J.; Riddle, S. R.; Kravchuk. A. V.: Zhao, Z.; Liao, H.; Bruzik, K. S.; Tsai, M.-D. “Phosphatidylinositol phospholipase C: kinetic and stereochemical evidence for an interaction between arginine-69 and the phosphate group of phosphatidylinositol”. Biochemistry (1997), 36(22), 6633-6642. 5. Sakharovsky, V. G.; Kravchuk. A. V.: Buzilova, I. G.; Kozlovsky, A. G. “5- Lactone of 2,3-anhydromevalonic acid is a novel metabolite of the alkaloid- producing strain Pénicillium sizovae." Prikl. Biokhim. Mikrobiol. (1994), 30(6), 794-8. 6. Litvinenko, L. A.; Petrikevich, S. B.; Kravchuk. A. V.: Grishchenkov, V. G.; Boronin, A. M. “Catabolism of caprolactam and its intermediates by industrial strains Pseudomonas putida BS394 containing various CAP plasmids.” Mikrobiologiya (1993), 62(3), 447-52 7. Litvinenko, L. A.; Kravchuk. A. V.: Petrikevich, S. B.; Sacharovsky, V. G.; Ivanitskaya, Ju. G.; Gulamova, D. E. “Influence of heavy water on growth, glucose assimilation, and stability of Escherichia coli to freezing-thawing” Mikrobiologiya (1992), 61(6), 1030-7. FIELDS OF STUDY Major Field: Chemistry VI TABLE OF CONTENTS Page Abstract.................................................................................................................................... ii Acknowledgments ................................................................................................................... iv Vita............................................................................................................................................V List of Tables..........................................................................................................................xii List of Figures .........................................................................................................................xiv Abbreviations ..........................................................................................................................xx Chapters: 1. Introduction .......................................................................................................1 I. I Mammalian PI-PLCs......................................................................................2 1.1.1 Structure............................................................................................... 2 1.1.2 Function and ..regulation......................................................................4 1.1.3 Mechanism............................................................................................ 8 1.2 Bacterial PI-PLC.............................................................................................10 1.2.1 Structure...............................................................................................10 1.2.2 Function and specificity .................................................................... 12 1.2.3 Mechanism.......................................................................................... 12 VII 1.2.4 Comparison with RNase A ................................................................ 15 NMR studies of bacterial PI-PLC .................................................................. 16 2.1 Introduction ...................................................................................................... 16 2.1.1 NMR of large proteins ........................................................................16 2.1.2 pH-activity profiles
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