Louisiana State University LSU Digital Commons LSU Historical Dissertations and Theses Graduate School 1987 Conformational Studies of Carnitine and Acetylcarnitine: Design and Synthesis of Active- Site Probes of Carnitine Acetyltransferase. William Jay Colucci Louisiana State University and Agricultural & Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_disstheses Recommended Citation Colucci, William Jay, "Conformational Studies of Carnitine and Acetylcarnitine: Design and Synthesis of Active-Site Probes of Carnitine Acetyltransferase." (1987). LSU Historical Dissertations and Theses. 4348. https://digitalcommons.lsu.edu/gradschool_disstheses/4348 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Historical Dissertations and Theses by an authorized administrator of LSU Digital Commons. 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Other _______________________________________________________________ _____ University Microfilms International Conformational Studies of Carnitine and Acetylcarnitine Design and Synthesis of Active-Site Probes of Carnitine Acetyltransferase A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements- for the degree of Doctor in Philosophy in The Department of Chemistry by William Jay Colucci B.A., Western Connecticut State College Danbury Connecticut, 1979 M a y , 1987 ACKNOWLEDGMENTS I would be amiss if I did not first thank my wife for bearing with me during the preparation of this manu­ script but, more importantly, for bearing down when the time was right. The work appearing in this manuscript was accom­ plished under the watchful eyes of Professor Richard D. Gandour. He guided me throughout, yet had the insight to let me explore. More importantly, he taught me that a chemist is Judged not by what he knows, but by what he does. My gratitude cannot be overstated. Next in line I wish to thank all of my fellow group members, especially John W. Nail, who showed me the vii— tues of cynicism in science; John C. Hogan, with whom I enjoyed many hours of metaphysical discussion and practi­ cal laughter; Drs. Julian Tirado-Rives and Mike A. Oli­ ver, from whom I gained an abundance of practical labora­ tory skills; and finally the newer members of the group, Gabriel (Gabby) • Garcia and Nabeel Bill Nabulsi, for assisting me in the preparation of a clean document. Throughout my research I have had the good fortune of instructing several very bright undergraduates; to them I am grateful for incalculable contributions. Spe­ cial thanks to: Terry C. Stelly, who synthesized the ii hemicholinium compounds and hemipalmitoylcarnitinium; H. Drew Poret, who assisted in the synthesis of (R)-sulfocarnitine; Stan Turnbull Jr., who recorded the IR spectra and optical rotations and helped break ground on the synthesis of sodium (R)-norcarnitine; and finally Mike (Don) Bruce, who prepared many valuable intermedi­ ates. I wish to thank Drs. Frank R. Fronczek and Steven F. Watkins for carefully determining the crystal structures appearing in this report; Mr. Marcus Nauman and Dr. Edward A. Mooberry for guiding me in my NMR wanderings; Dr. David Coy of Tulane Medical Center for allowing us the use of his micropolarimeter; and Mr. Robert R. Zinn for helping me to implement many of the forqe field cal­ culations. I wish also to thank Dr. Aksel A. Bothnei— By of Carnegie-Mellon University for his invaluable communi­ cations, which increased immensely our understanding of the carnitine and acetylcarnitine proton spectra. A very special acknowledgment goes to Drs. Linda and Paul Brady (The Brady Bunch) for their invaluable collaborative effort in assaying the compounds which we synthesized. In addition, I would like to express my appreciation to the members of ray examining committee; Professors Ezzat S. Younathan, James G. Traynham, Robert J. Gale, Neil R. Kestner, and Michael E. Orlowski, for their many in helpful suggestions, and corrections to an earlier vei— sion of this document; also to the Charles E. Coates Mem­ orial Fund of the LSU Foundation (donated by Dr. George H. Coates), for financial provision in the production of this dissertation. Finally, I would like it known that my life has been enriched for having known and worked with the following good friends and colleagues: Dr. Rayomand J. Unwalla, Dr. Raymond Piccione, and Hessara Ghane; their philosophical and political discussions I found immensely enjoyable. Many others, both student and faculty, to whom I owe a debt of thanks, go unnamed but not unappreciated; they know who they are. Williai Colucci iv To Mary J. and Edward E. in memoriam and to Trudy and Jamie Lynn v TABLE OF CONTENTS ACKNOWLEDGMENTS .........................................ii DEDICATION .............................................. v TABLE OF CONTENTS ...................................... vi LIST OF TABLES .......................................... xii LIST OF FIGURES .............. xvi ABSTRACT .................................................xix LIST OF ABBREVIATIONS .................................. xxi INTRODUCTION ............................................ xxii References ........................................xxvii CHAPTER 1. A Review of Carnitine and Proteins That Bind Carnitine as a Substrate: Catalysis by Carnitine Acetyltransferase ................. 1 Discovery and Function of Carnitine ......2 Carnitine Specific Proteins .................... 5 Carnitine active transport Carnitine-acylcarnitine translocase Acylcarnitine hydrolase Carnitine acyltransferases Distribution of CAT Distribution of COT Inhibitors of COT Distribution of CPT Inhibitors of CPT The Carnitine Acyl Transport System ............22 Biochemical Characterization of CAT ............24 Reaction parameters Kinetic mechanisms Enzyme ORD spectrum Inhibition by (S)-isomers pH Dependence Inhibition by dications Sulfhydryl-specific reagents CoA analogues Effect of chain length v| Suicide substrate inhibitors Competitive carnitine analogues Alternative substrates Mechanisms of catalysis Summary References ........... 59 CHAPTER 2. Crystal Structures of Carnitine and Acetylcarnitine Zwitterions: A Structural Hypothesis for Mode of Action ................. 66 Introduction ....................................... 67 Experimental Procedures ...........................68 Materials Diffraction data Structure solution and refinement Results and Discussion ............................70 Crystal structures Comparison with previous work Intramolecular electrostatic attraction question Explanation of conformational preference Hypothesis for conformer selective binding in carnitine acetyltransferase Acknowledgments ....................................77 References ......................................... 77 CHAPTER