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MECHANISM of the REACTION CATALYZED by the OXALATE DECARBOXYLASE from Bacillus Subtilis

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MECHANISM of the REACTION CATALYZED by the OXALATE DECARBOXYLASE from Bacillus Subtilis MECHANISM OF THE REACTION CATALYZED BY THE OXALATE DECARBOXYLASE FROM Bacillus subtilis By DRAŽENKA SVEDRUŽIĆ A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2005 Copyright 2005 by Draženka Svedružić This thesis is dedicated to my brother Željko, my parents and Chris. Ova je teza posvećena mome bratu Željku, mojim roditeljima i Krisu. ACKNOWLEDGMENTS This study was supported by grants from the National Institutes of Health (DK61666 and DK53556) and by the University of Florida Chemistry Department. Partial funding was also received from Dr. Ammon B. Peck. Thanks go to my doctoral dissertation committee: Dr. Steven A. Benner, Dr. Ammon B. Peck, Dr. Michael J. Scott, Dr. Jon D. Stewart, and especially my advisor, Dr. Nigel G. J. Richards, for making this research project a reality, but also for his constant support and guidance. Dr. Laurie A. Renhardt, Yang Liu and Dr. Wallace W. Cleland I thank for fruitful collaboration on heavy atom isotope effects research. Also, I thank Dr. Wallace W. Cleland for hospitality during my stay in Madison, Wisconsin. Thanks go to my EPR collaborators Dr. Lee Walker, Dr. Andrzej Ozarowski and Dr. Alexander Angerhofer. I am grateful to my coworkers and friends in the Richards research group, especially Dr. Christopher H. Chang for proofreading, countless discussions, valuable insights, guidance and support. Special thanks go to Stefan Jonsson for all his help, Sue Abbatiello for her mass spectrometry efforts and Lukas Koroniak for help with NMR experiments. Also thanks go to all members of Richards group, especially Mihai, Jemy and Cory, for providing a pleasant and supporting environment in and out of lab. I thank Dr. James A. Deyrup, Graduate Coordinator, and Lori Clark in the Graduate Student Office for their help and advice. iv Finally, I thank my mother, father and my brother for all their support and their sacrifice that enabled me to get high education. Also, thanks go to my brother Željko for guidance and inexhaustible inspiration. v TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................................................................................. iv LIST OF TABLES........................................................................................................... viii LIST OF FIGURES ........................................................................................................... ix ABSTRACT...................................................................................................................... xii CHAPTER 1 INTRODUCTION ........................................................................................................1 Biological Significance of Oxalic Acid........................................................................1 Oxalate-Degrading Enzymes........................................................................................3 Oxalate Decarboxylase .................................................................................................4 Biological Distribution ..........................................................................................4 Induction and Biological Roles .............................................................................5 Enzyme Structure: OXDCase Belongs to Bicupin Structural Family...................6 Enzyme –Manganese Interaction ..........................................................................9 Enzyme-Oxygen Interaction and Production of Peroxide...................................12 Chemical Degradation of Oxalate ..............................................................................14 Research Objective .....................................................................................................15 2 EXPRESSION AND PURIFICATION OF RECOMBINANT B. subtilis OXALATE DECARBOXYLASE..............................................................................16 Introduction.................................................................................................................16 Results and Discussion ...............................................................................................17 Experimental Section..................................................................................................21 3 STEADY STATE CHARACTERISATION OF THE REACTION CATALYZED BY B. subtilis OXALATE DECARBOXYLASE......................................................26 Introduction.................................................................................................................26 Results and Disscusion ...............................................................................................27 Progress Curve and Michaelis-Menten Equation................................................27 Activation, Inhibition and Substrate Specificity .................................................30 The pH Dependence on OxdC Catalysis.............................................................33 vi Formate Production: Irreversible or Reversible Process.....................................36 Experimental Section..................................................................................................37 4 HEAVY ATOM ISOTOPE EFFECTS ON THE REACTION CATALYZED BY B. subtilis OXALATE DECARBOXYLASE.............................................................39 Introduction.................................................................................................................39 Results and Discussion ...............................................................................................41 13C and 18O Isotope Effects .................................................................................41 Kinetic Mechanism for the Enzymatic Reaction of OxdC..................................43 Fractionation Factors...........................................................................................44 Molecular Mechanism of the Reaction Catalyzed by OxdC...............................50 Experimental Section..................................................................................................53 5 ROLES OF THE ACTIVE SITE ARGININE AND GLUTAMATE RESIDUES....57 Introduction.................................................................................................................57 Results and Discussion ...............................................................................................61 Mutational Effects on OxdC Structure................................................................61 Steady-State Characterization .............................................................................62 Oxalate Oxidase and Dye Oxidation Activities...................................................65 13C and 18O Kinetic Isotope Effects.....................................................................68 Experimental Section..................................................................................................72 6 IDENTIFICATION AND STUDIES OF TYROSYL RADICAL FORMED DURING TURNOVER..............................................................................................74 Introduction.................................................................................................................74 Results and Discussion ...............................................................................................76 Variation of Radical Intensity as a Function of Time .........................................76 Effect of Temperature and Microwave Power on Radical Signal.......................79 Identification of a Tyrosyl Radical......................................................................83 In Search for Tyrosine Residues Forming the Radical........................................83 Spin-trapping experiments...................................................................................86 Potential Functional Implications of the Observed Radical................................87 Experimental Section..................................................................................................89 APPENDIX A PROTEIN PURIFICATION CHROMATOGRAMS.................................................94 B DERIVATION OF EQUATIONS FOR HEAVY ATOM ISOTOPE EFFECT ........97 C EPR SPIN-TRAPPING OF A RADICAL DURING OXALATE DECARBOXYLASE TURNOVER.........................................................................100 BIOGRAPHICAL SKETCH ...........................................................................................109 vii LIST OF TABLES Table page 2-1 Purification table for a typical OxdC preparation. ...................................................19 3-1 Specific activities (SA) and Michaelis-Menten constants (KM) for oxalate decarboxylation catalyzed by native and recombinant OXDCase ...........................29 3-2 Specific activities and kinetic constants: kcat, KM and kcat/KM for oxalate decarboxylation catalyzed by recombinant OxdC. ..................................................30 3-3 Results of the deuterium exchange experiments......................................................37 4-1 13C and 18O isotope effects (IE) on the reaction catalyzed by Bacillus subtilis.OxdC............................................................................................................41 5-2 Molecular weights (MW) of OxdC in solution and manganese incorporation per momomer. ................................................................................................................62
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