Mechanistic Studies of Two Selected Flavin-Dependent Enzymes: Choline Oxidase and D-Arginine Dehydrogenase
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Georgia State University ScholarWorks @ Georgia State University Chemistry Dissertations Department of Chemistry Summer 8-11-2011 Mechanistic Studies of Two Selected Flavin-Dependent Enzymes: Choline Oxidase and D-Arginine Dehydrogenase Hongling Yuan Georgia State University Follow this and additional works at: https://scholarworks.gsu.edu/chemistry_diss Part of the Chemistry Commons Recommended Citation Yuan, Hongling, "Mechanistic Studies of Two Selected Flavin-Dependent Enzymes: Choline Oxidase and D- Arginine Dehydrogenase." Dissertation, Georgia State University, 2011. https://scholarworks.gsu.edu/chemistry_diss/56 This Dissertation is brought to you for free and open access by the Department of Chemistry at ScholarWorks @ Georgia State University. It has been accepted for inclusion in Chemistry Dissertations by an authorized administrator of ScholarWorks @ Georgia State University. For more information, please contact [email protected]. MECHANISTIC STUDIES OF TWO SELECTED FLAVIN-DEPENDENT ENZYMES: CHOLINE OXIDASE AND D-ARGININE DEHYDROGENASE by HONGLING YUAN Under the Direction of Giovanni Gadda ABSTRACT Choline oxidase catalyzes the flavin-dependent, two-step oxidation of choline to glycine betaine via the formation of an aldehyde intermediate. The oxidation of choline includes two reductive half-reactions followed by oxidative half-reactions. In the first oxidation reaction, the alcohol substrate is activated to its alkoxide via proton abstraction and oxidized via transfer of a hydride from the alkoxide α-carbon to the N(5) atom of the enzyme-bound flavin. In the wild- type enzyme, proton and hydride transfers are mechanistically and kinetically uncoupled. The role of Ser101 was investigated in this dissertation. Replacement of Ser101 with threonine, alanine, cysteine, or valine demonstrated the importance of the hydroxyl group of Ser101 in proton abstraction and in hydride transfer. Moreover, the kinetic studies on the Ser101Ala variant have revealed the importance of a specific residue for the optimization of the overall turnover of choline oxidase. The UV-visbible absorbance of Ser101Cys suggests Cys101 can form an adduct with the C4a atom of the flavin. The mechanism of formation of the C4a- cysteinyl adduct has been elucidated. D-arginine dehydrogenase (DADH) catalyzes the oxidation of D-amino acids to the corresponding imino acids, which are non-enzymatically hydrolyzed to α-keto acids and ammonia. The enzyme is strick dehrogenase and deoesnot react with molecular oxygen. Steady state kinetic studies wirh D-arginine and D-histidine as a substrate and PMS as the electron acceptor has been investigated. The enzyme has broad substrate specificity for D-amino acids except aspartate, glutamate and glycine, with preference for arginine and lysine. Leucine is the slowest substrate in which steady state kinetic parameters can be obtained. The chemical mechanism of leucine dehydrogenation catalyzed by DADH was explored with a combination of pH, substrate and solvent kinetic isotope effects (KIE) and proton inventories by using rapid kinetics in a stopped-flow spectrophotometer. The data are discussed in the context of the crystallographic structures at high resolutions (<1.3 Å) of the enzyme in complex with iminoarginine or iminohistidine. INDEX WORDS: Choline oxidase, Hydroxyl group, C4a-cysteinyl adduct, D-arginine dehydrogenase, Conformational change, Hydride transfer MECHANISTIC STUDIES OF TWO SELECTED FLAVIN-DEPENDENT ENZYMES: CHOLINE OXIDASE AND D-ARGININE DEHYDROGENASE by HONGLING YUAN A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the College of Arts and Sciences Georgia State University 2011 Copyright by Hongling Yuan 2011 MECHANISTIC STUDIES OF TWO SELECTED FLAVIN-DEPENDENT ENZYMES: CHOLINE OXIDASE AND D-ARGININE DEHYDROGENASE by HONGLING YUAN Committee Chair: Dr. Giovanni Gadda Committee: Dr. Al Baumstark Dr. Jenny J. Yang Electronic Version Approved by: Office of Graduate Studies College of Arts and Sciences Georgia State University August 2011 iv ACKNOWLEDGMENTS Five years of Ph.D. life will end with writing of the dissertation, but in my life it is just a comma. I will face the beginning of another journey. This dissertation would not have been possible without the help, support, guidance and efforts of a lot of people because of whom my graduate experience has been one that I will cherish forever. I would like to take this opportunity to express my gratitude to them for completing the dissertation successfully. My deepest gratitude is to my advisor, Dr. Giovanni Gadda, for his excellent guidance, encouragement, caring, supervising and providing me with an excellent atmosphere for doing research. His encouragement made me feel confident to fulfill my desire and to overcome every difficulty I encountered. During my studies, he taught me not only the enzymology knowledge but also his stringent academic attitude to be a good scientist. Until now I just realize without his “push”, I can never improve myself. I cannot say thank you enough for his tremendous support and help. I am grateful to my committee members, Dr. Baumstark and Dr. Yang, for their encouragement, practical advice and challenge throughout my academic program. Dr. Baumstark’s encouraging words inspire me to approach my work as a social scientist. Dr. Yang, I would like to thank you for your motherly care in my work and my personal life. Without their support, I could not have done what I was able to do. I am also grateful to the all the professors for their advice and teaching during my Ph.D. studies. I would also like to thank members past and present in Dr. Giovanni Gadda’s group: Osbourn, Steffan, Kunchala, Slavica, Kevin, Tran Bao, Merid, Andrea, Stephen, Danila, Sharonda, Lydia, Philip, Swathi, Crystal, Rizvan and Sandra for sharing their enthusiasm and comments on my work. It would have been a lonely lab without them. Osbourne, you are the v first labmate I met, I am so glad to have you and I still remember you kindly teach me how to pronounce English. To Slavica, thank you for encouraging me to be a strong woman. I really enjoy the drinking break and conversation about life. I thank Swathi for helping me with the tone and my writing. I really had fun in the step class we took. I have been especially fortunate to have Andrea and Kunchala during my PhD studies. I cannot find any words to express my thanks. They are like my brother and sister. Without Kunchala, I cannot get used to the abroad life so easily. Every time when I have problem I know you are there and you help me. Andrea, thank- you for your patience and time to listen to my complaints. You have always listened when I needed to talk. The care, discipline and organization that you bring to this work have had a beneficial effect on my work. I am also thankful to the following former or current staff at GSU: Edna, Sina, Diane, Will, Chris, Dan, Stephanie, Robert, Don Hardon etc. your generous and timely help and various forms of support make my graduate studies easy and enjoyable. I’d like to convey my heartfelt thanks to my forgiving, generous and loving friends. They have helped me stay sane and stay focused on my graduate study when I am depressed. Their care and patience on teaching me driving and cooking helped me overcome the difficulties I met in the US and without them I could not have survived the process. I greatly value their friendship and deeply appreciate their help in my personal life. Most importantly, I would also like to thank my mom, who has always supported, encouraged and believed in me. My brothers and sisters, their understanding and love encouraged me to work hard and to continue pursuing a Ph.D. project abroad. They always give their best wishes to me. Finally, I appreciate the financial support from NSF that funded the research discussed in this dissertation. vi TABLE OF CONTENTS ACKNOWLEDGMENTS ............................................................................................................. iv LIST OF TABLES ...........................................................................................................................x LIST OF FIGURES ....................................................................................................................... xi LIST OF SCHEMES......................................................................................................................xv CHAPTER 1 ..................................................................................................................................17 Introduction ....................................................................................................................................17 1.1 Flavoproteins ........................................................................................................................ 17 1.2 Mechanisms of Oxidation of Carbon-Heteroatom Bond ...................................................... 19 1.3 Protein Hydroxyl Group Proximal to the C(4a)-N(5) Flavin Atoms .................................... 24 1.3.1 Physical Properties of Hydroxyl Group .............................................................. 24 1.3.2 Conserved Hydroxyl Group in Flavoenzymes .................................................... 25 1.4 Specific Goals ....................................................................................................................... 37 1.5 References ...........................................................................................................................