University of Kentucky UKnowledge University of Kentucky Doctoral Dissertations Graduate School 2006 STRUCTURAL AND FUNCTIONAL STUDIES OF SYNAPTIC ENZYMES Eun Jeong Lim University of Kentucky, [email protected] Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Lim, Eun Jeong, "STRUCTURAL AND FUNCTIONAL STUDIES OF SYNAPTIC ENZYMES" (2006). University of Kentucky Doctoral Dissertations. 259. https://uknowledge.uky.edu/gradschool_diss/259 This Dissertation is brought to you for free and open access by the Graduate School at UKnowledge. It has been accepted for inclusion in University of Kentucky Doctoral Dissertations by an authorized administrator of UKnowledge. For more information, please contact [email protected]. ABSTRACT OF DISSERTATION Eun Jeong Lim Department of Molecular and Cellular Biochemistry College of Medicine University of Kentucky 2006 STRUCTURAL AND FUNCTIONAL STUDIES OF SYNAPTIC ENZYMES ABSTRACT OF DISSERTATION A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Molecular and Cellular Biochemistry and College of Medicine at the University of Kentucky By Eun Jeong Lim Lexington, Kentucky Director: Dr. David W. Rodgers Associate Professor of Molecular and Cellular Biochemistry Lexington, Kentucky 2006 Copyright © Eun Jeong Lim, 2006 ABSTRACT OF DISSERTATION STRUCTURAL AND FUNCTIONAL STUDIES OF SYNAPTIC ENZYMES Thimet oligopeptidase (TOP, EC 3.4.24.15) and neurolysin (EC 3.4.24.16) are zinc dependent metallopeptidases that metabolize small bioactive peptides. The two enzymes share 60 % sequence identity and their crystal structures demonstrate that they adopt nearly identical folds. They generally cleave at the same sites, but they recognize different positions on some peptides, including neurotensin, a 13-residue peptide involved in modulation of dopaminergic circuits, pain perception, and thermoregulation. On the basis of crystal structures and previous mapping studies, four residues (E469/R470, M490/R491, H495/N496, and R498/T499, TOP residues listed first) in the substrate-binding channel appear positioned to account for differences in specificity. TOP mutated to the neurolysin residues at all four position cleaves neurotensin at the neurolysin site and neurolysin mutated to the TOP residues at all four sites cleaves at the TOP position. Using a series of constructs mutated at only three sites, it was determined that only two of the mutations, E469/R470 and R498/T499, are required to swap the specificity of TOP and neurolysin. These results were confirmed by testing the two mutation constructs, and either single mutant of TOP shown an intermediate specificity, cleaving at both sites. Crystal structures of the two mutation constructs of TOP and neurolysin unliganded forms, the mutations do not perturb local structure, but side chain conformations at the R498/T499 position differ from those of the mimicked enzyme. A model for differential recognition of neurotensin based on differences in surface charge distribution in the substrate binding sites is proposed. The model is supported by finding that reducing the positive charge on the peptide results in cleavage at both hydrolysis sites. This dissertation also includes a description of the production and crystallization trials of human neprilysin (E.C. 3.4.24.11), which will be used as another model system for studying specificity in metallopeptidases. In addition, the production and crystallization, and crystal characterization of human choline acetyltransferase (EC 2.3.1.6) is described. KEYWORDS: Thimet oligopeptidase, neurolysin, crystallography, neurotensin, specificity STRUCTURAL AND FUNCTIONAL STUDIES OF SYNAPTIC ENZYMES By Eun Jeong Lim Director of Dissertation Director of Graduate Studies Date RULES FOR THE USE OF DISSERTATIONS Unpublished dissertations submitted for the Doctor’s degree and deposited in the University of Kentucky Library are as a rule open for inspection, but are to be used only with due regard to the rights of the authors. Bibliographical references may be noted, but quotations or summaries of parts may be published only with the permission of the author, and with the usual scholarly acknowledgements. Extensive copying or publication of the dissertation in whole or in part also requires the consent of the Dean of the Graduate School of the University of Kentucky. A library that borrows this dissertation for use by its patrons is expected to secure the signature of each user. DISSERTATION Eun Jeong Lim The Graduate School University of Kentucky 2006 STRUCTURAL AND FUNCTIONAL STUDIES OF SYNAPTIC ENZYMES DISSERTATION A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Molecular and Cellular Biochemistry and College of Medicine at the University of Kentucky By Eun Jeong Lim Lexington, Kentucky Director: Dr. David W. Rodgers Associate Professor of Molecular and Cellular Biochemistry Lexington, Kentucky 2006 Copyright © Eun Jeong Lim, 2006 Dedicated to my parents, Jong Man Lim and Won Ja Hong – who are my role models. Their enthusiastic life, hard working, and moral characters always inspire me. ACKNOWLEDGEMENTS I would like to express my gratitude to my research mentor and dissertation advisor, Associate Professor David Rodgers, for his guidance, patience, and encouragement throughout my graduate studies. Dr. Rodgers provided me theoretical aspects of x-ray crystallography and taught me how to determine the crystal structures of macromolecules. And he always finds the time for listening to the little problems and roadblocks that unavoidably crop up in the course of performing research. His technical and theoretical advice was necessary for the completion of this dissertation and has taught me valuable lessons and insights on the workings of academic research in general. I am also grateful to my dissertation committee members, Dr. Thomas Vanaman, Dr. Rebecca Dutch, Dr. Charles Snow, and Dr. Robert Houtz for reading previous drafts of this dissertation and providing many valuable comments that improved the presentation and contents of this dissertation. I am also indebted to my former advisor, Dr. Young Gyu Chai. He always listened to my concerns and gave a lot of good advice even when I was preparing to study abroad and when I was determining the lab. And I am also thankful to Dr. Myung Hee Kim for her friendship, support, and encouragement. I would also like to thank present members of the Dr. Rodgers’ lab: Dr. Jack Schmidt for helping to determine kinetics of my proteins, Sowmya Sampath, Nick Noinaj for helping me how to use a lot of software, and Jerry Coll-Rodriguez for helping to prepare proteins. And I also thankful to former lab members, Dr. Christina Hines, Dr. Yiying Cai, Dr. Kallol Ray, and Dr. Chad Haynes, for their friendship and support. Finally, and most importantly, I would like to thank my husband Jun Ho Roh for his love and understanding during the past few years. I could not finish this dissertation without his support, patience, and encouragement. I also thank my parents, Jong Man Lim and Won Ja Hong, my sisters, Eun Suk and Eun Hee, and my brother, Jeong Hun, for their faith in me and providing me with unending encouragement and support. iii TABLE OF CONTENTS Acknowledgements........................................................................................................................iii List of Tables ................................................................................................................................. vi List of Figures...............................................................................................................................vii List of Files .................................................................................................................................... ix Chapter One: Introduction .............................................................................................................. 1 Neuropeptides and neuropeptidases.................................................................................... 1 Substrate specificity in neuropeptidases (fuzzy specificity)............................................... 2 Thimet oligopeptidase (TOP) and neurolysin..................................................................... 4 Neurotensin......................................................................................................................... 8 The present study ................................................................................................................ 9 Significance....................................................................................................................... 10 Neprilysin.......................................................................................................................... 11 Human choline acetyltransferase (hChAT) ...................................................................... 13 Chapter Two: Materials and methods........................................................................................... 22 Preparation of TOP and neurolysin mutant constructs ..................................................... 22 Expression and purification of wild type TOP and mutants............................................. 23 Expression and purification of wild type neurolysin and mutants.................................... 24 Assay for cleavage