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Utah State University DigitalCommons@USU All Graduate Theses and Dissertations Graduate Studies 5-2013 Structural and Functional characterization of the LDP3 (VHZ) Protein Tyrosine Phosphatase Vyacheslav I. Kuznetsov Utah State University Follow this and additional works at: https://digitalcommons.usu.edu/etd Part of the Organic Chemistry Commons Recommended Citation Kuznetsov, Vyacheslav I., "Structural and Functional characterization of the LDP3 (VHZ) Protein Tyrosine Phosphatase" (2013). All Graduate Theses and Dissertations. 2027. https://digitalcommons.usu.edu/etd/2027 This Dissertation is brought to you for free and open access by the Graduate Studies at DigitalCommons@USU. It has been accepted for inclusion in All Graduate Theses and Dissertations by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF THE LDP3 (VHZ) PROTEIN TYROSINE PHOSPHATASE by Vyacheslav I. Kuznetsov A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Chemistry (Organic Chemistry) Approved: ____________________ ____________________ Dr. Alvan C. Hengge Dr. Bradley S. Davidson Major Professor Committee Member ____________________ ____________________ Dr. Sean J. Johnson Dr. Daren P. Cornforth Committee Member Committee Member ____________________ ____________________ Dr. Lisa M. Berreau Dr. Mark R. McLellan Committee Member Vice President for Research and Dean of the School of Graduate Studies UTAH STATE UNIVERSITY Logan, Utah 2013 ii Copyright © Vyacheslav I. Kuznetsov 2013 All Rights Reserved iii ABSTRACT Structural and Functional Characterization of the LDP3 (VHZ) Protein Tyrosine Phosphatase by Vyacheslav I. Kuznetsov, Doctor of Philosophy Utah State University, 2013 Major Professor: Alvan C. Hengge Department: Chemistry and Biochemistry PTPs represent a large family of enzymes responsible for the intracellular dephosphorylation. Together with protein tyrosine kinases (PTKs), PTPs control the level of protein phosphorylation which modulates numerous aspects of cell life. VHZ, a recently discovered PTP, expresses in most fetal tissues and is associated with several types of cancer. It was originally classified as a dual specificity phosphatase (DUSP23) and named after the prototypical member as VH1 related protein member Z. Our preliminary screening assays of VHZ performed with 360 phosphopeptides have revealed a number of phosphotyrosine containing substrates, but no activity toward phosphothreonine or phosphoserine substrates, thus strongly suggesting that this enzyme is not related to DUSPs. We solved a crystal structure to 1.1 Å resolution, which, together with sequence analysis showed that VHZ is more related to classical PTPs rather than DUSPs. The crystal structure revealed an unusual metavanadate (VO3) ion in the iv active site of VHZ which has never been observed in the aqueous environment. Its appearance in our crystal structure, together with computational studies, allowed us to propose a mechanism by which PTPs can enzymatically interconvert different non- inhibiting vanadate species dominating at physiological pH to generate the inhibiting metavanadate ion observed in our structure. We also evaluated VHZ catalytic properties by several techniques including steady-state and pre-steady-state kinetics, kinetic isotope effects, kinetic inhibition studies combined with site-directed VHZ modification, and computer modeling. We found that the catalytic activity of VHZ was significantly underestimated by previous researchers. Unlike any other PTP, VHZ has evolved with two general acids in the active site – a phenomenon that was expedited by two substrate binding orientations. Finally, we show that VHZ is capable of performing hydrolysis as well as alcoholysis of its phosphate monoester substrates. We were able to identify a single mutation in the area of the active site responsible for this unusual phosphotransferase behavior. Considering that the level of intracellular phosphorylation affects a number of normal and pathological physiological processes, our finding may explain the relation of VHZ to several types of cancer and justify this enzyme as a promising drugable target. (219 pages) v PUBLIC ABSTRACT Structural and Functional Characterization of the LDP3 (VHZ) Protein Tyrosine Phosphatase by Vyacheslav I. Kuznetsov Utah State University, 2013 Protein phosphorylation is as an important regulatory mechanism that controls a large number of physiologically important cellular processes. Proteins are phosphorylated on aliphatic (serine and threonine) as well aromatic (histidine and tyrosine) residues predominantly located on sterically accessible loops. Phosphorylation affects three- dimensional structures of proteins resulting in conformational changes which activate or deactivate enzymatic functions. Because of the ability of protein phosphorylation to modulate a multitude of diverse cellular processes, it became an appealing and promising target for drug therapy. Attachment of phosphate is accomplished by kinases, whereas the removal of the phosphoryl group is performed by protein phosphatases. Traditionally, protein kinases have been studied more intensely than protein phosphatases due to the earlier belief that they confer fine regulation to protein phosphorylation, whereas protein phosphatases act to nonspecifically remove phosphate groups. In recent years the protein phosphatases were subjected to thorough analysis to reveal a number of diverse properties which formed the basis for phosphatase classification. The family of protein tyrosine phosphatses (PTPs) represents the largest group of protein phosphatases known today. We focused our study on the 150 amino acids VHZ phosphatase, which was originally classified as a dual-specificity enzyme which can dephosphorylate aliphatic (phospho serine, phospho threonine) and aromatic (phospho tyrosine) residues. We solved a crystal structure of VHZ refined to 1.1 Å resolution and performed an extensive structural, biological and kinetic analysis which revealed that this enzyme is not a dual specificity phosphatase. Activity screening with 360 biologically relevant peptides indicated strict phospho-tyrosine specificity and provided important information about its physiological substrates. Mechanistic studies using steady-state and pre-steady state kinetics, as well as inhibition studies and kinetic isotope effects (KIE) explain the significantly reduced catalytic parameters of VHZ, and contribute to our understanding of the PTP catalytic mechanism in general. vi ACKNOWLEDGMENTS First of all, I would like to thank my major professor, Dr. Alvan C. Hengge, for accepting me in his laboratory and entrusting me with outstanding research projects which yielded over the past few years several publications presented in this dissertation. His mentorship and generous support gave me an outstanding opportunity to pursue my long-lasting goals of becoming a successful and independent researcher. I would like to thank my supervisory committee Drs. Alvan C. Hengge, Sean J. Johnson, Bradley S. Davidson, Lisa M. Berreau, and Daren P. Cornforth, who have been incredibly supportive. Their valuable suggestions helped me to progress through my academic years and were outstandingly helpful in many challenging research situations. I would like to thank to the former and present members of the Hengge lab, especially Drs. Mark Haney and Tiago Brandao, with whom we have eaten barrels of salt while traveling along the winding road of scientific discoveries. They were extremely motivating and helpful, always willing to share their own knowledge and experience, and they provided a valuable expertise and critical thinking in many aspects of my research project. I would like to give special thanks to Margaret Dobrowolska. Her knowledge, attention, and motherly care were absolutely invaluable and guided me from the first moment I applied to enter USU PhD program up to the moment of my defense and graduation. vii I would like to thank USU and the Department of Chemistry and Biochemistry for giving the opportunity to study, and to participate in scientific meetings and providing all the equipment needed to conduct my research. Lastly I would like to thank my mother and my sister for their patience, understanding, and moral support over these lasting years of separation. Thank you everyone! Vyacheslav I. Kuznetsov viii In the memory of my grandfather, for his invaluable support, directions and kindness which I will never get a chance to repay ix CONTENTS Page ABSTRACT.......................................................................................................................iii PUBLIC ABSTRACT.........................................................................................................v ACKNOWLEDGMENTS..................................................................................................vi LIST OF TABLES...............................................................................................................x LIST OF FIGURES............................................................................................................xi LIST OF SCHEMES........................................................................................................xiv LIST OF ABBREVIATIONS............................................................................................xv CHAPTER 1. INTRODUCTION.......................................................................................1
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