Virginia Commonwealth University VCU Scholars Compass Theses and Dissertations Graduate School 2009 VITAMIN B6 METABOLISM AND REGULATION OF PYRIDOXAL KINASE Amit Gandhi Virginia Commonwealth University Follow this and additional works at: https://scholarscompass.vcu.edu/etd Part of the Chemicals and Drugs Commons © The Author Downloaded from https://scholarscompass.vcu.edu/etd/2008 This Dissertation is brought to you for free and open access by the Graduate School at VCU Scholars Compass. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of VCU Scholars Compass. For more information, please contact [email protected]. © Amit K. Gandhi 2009 All Rights Reserved VITAMIN B 6 METABOLISM AND REGULATION OF PYRIDOXAL KINASE A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University. By AMIT K. GANDHI M.S (Pharmaceutical Science), Rajiv Gandhi University, Indore, India, 2003 B.Pharm, Rajiv Gandhi University, Indore, India, 2001 Director: Martin K. Safo, Ph.D Assistant Professor, Department of Medicinal Chemistry Virginia Commonwealth University Richmond, Virginia December, 2009 Acknowledgement I would like to take this opportunity to express my deep gratitude and profound respect to my advisor, Dr. Martin K. Safo, for his supervision, advice, and guidance in this research work. His support and insight have been invaluable in the progression of my research. I also appreciate his words of encouragement, which kept me always in an innovative mood and guided me at all the times to bring about the best in me. He is my mentor and teacher whom I shall remember forever. I sincerely acknowledge Dr. Jason P. Rife, Dr. Glen E. Kellogg, Dr. Darrell L. Peterson and Dr. Walter M. Holmes for serving as my graduate committee members and for their advice, supervision, and support throughout my research. I extend my sincere thanks to Dr. Faik N. Musayev for teaching me various protein purification and crystallographic techniques. My heartfelt thanks go to Dr. Mohini S. Ghatage, who helped me in protein expression, purification, and enzyme kinetic studies. I would like to thank Dr. Richmond Danso-Danquah, who helped me in synthesis studies. iii I gratefully acknowledge Dr. Verne Schirch for his constant guidance and motivation throughout my research. I would also like to convey my regards and thanks to Dr. Donald J. Abraham, for the financial support provided to me. I can not express my emotions to my family, my wife, my parents, and my brother, who have provided countless moments of inspiration and motivation to achieve my goals. I also owe thanks to my all friends and all my well wishers, who have always inspired me for success. I also thank Institute for Structural Biology and Drug Discovery (ISBDD) for providing me all the necessary facilities and resources. Finally, I am grateful to the Department of Medicinal Chemistry, School of Pharmacy for giving me teaching assistantship and opportunity to perform research work. iv Table of Contents Page Acknowledgements............................................................................................................iii List of Tables .................................................................................................................... ix List of Figures ..................................................................................................................... x Abstract…….. .................................................................................................................. xiv Chapter 1 General Introduction .............................................................................................. 1 1.1 Pathways of Vitamin B 6 Metabolism…………………………………...1 1.2 Structure and Function of Pyridoxal Kinase…………………………... 3 1.3 Catalytic Mechanism of Pyridoxal Kinase …………………………... 4 1.4 Pyridoxine 5’-Phosphate Oxidase …………………………………….. 5 1.5 Vitamin B 6 Requiring Enzymes……………………………………….. 6 1.6 Vitamin B 6 Enzymes, Neurotransmitters and Brain Function………….8 1.7 PLP and Vitamin B 6 Toxicity ………………………………………… 9 1.8 MgATP Substrate Inhibition of PL Kinase in the presence of PLP ......10 1.9 Vitamin B 6 Deficiency ………………………………………………...10 1.10 PL Kinase Inhibitors and PLP Deficiency……………….…………...11 1.11 Genetic Defects in PL Kinase or PNP Oxidase and PLP Deficiency. .12 1.12 Other Disorders Associated with Vitamin B 6 Deficiency…………... 13 v 1.13 Conclusion……………………………..……………………………. 14 2 Determine the Kinetic and Structural Properties of Human PL Kinase………… 16 2.1 Introduction…………………………………………………………... 16 2.2 Methods 2.2.1 Expression and Purification of Human PL Kinase………………… 17 2.2.2 Determination of Kinetic Constants of PL and MgATP…………… 18 2.2.3 Human PL kinase Activity in the Presence of Monovalent Cations.. 18 2.2.4 Crystallization and Structure Determination of Human PL Kinase… 18 2.3 Results and Discussion 2.3.1 Kinetic Constants and Synergism Between PL and ATP…………… 22 2.3.2 Effect of Different Monovalent Metals on Human PL Kinase activity…………………………………………………… 26 2.3.3 Structural Studies……………………………………………………. 26 2.4 Conclusion…………………………………………………………….. 34 3 Determine the Role of the Active site Residues Asp235 in PL Kinase Catalytic Function ………………………………………………………….…… 36 3.1 Introduction…………………………………………………………… 36 3.2 Methods 3.2.1 Site-directed Mutagenesis and purification of enzymes……………. 37 3.2.2 Determination of Kinetic Constants ………………………………... 38 vi 3.2.3 Crystallization, Data Collection, and Structure Determination ……. 38 3.3 Results and Discussion………………………………………………. 42 3.4 Conclusion……………………………………………………………. 48 4 Determine the Method of Kinetic Regulation of PL Kinase … ……………… 49 4.1 Introduction…………………………………………………………… 49 4.2 Methods 4.2.1 MgATP Substrate Inhibition Studies ……………………………… 50 4.2.2 ADP Product Inhibition Studies …………………………………..... 51 4.2.3 Crystallization, Data Collection, and Structure Determination ……..51 4.3 Results and Discussion 4.3.1 Severe MgATP Substrate Inhibition of PL Kinase in the Presence of PLP and Evidence of Tight PLP Binding in PL Kinase……….. 51 4.3.2 ADP Product Inhibition of PL Kinase……………………………… 57 4.3.3 MgATP and PLP Bind to PL Kinase to Form a Ternary complex…. 57 4.4 Conclusion …………………………………………………………… 62 5 Identify Drugs and Other Compounds that are Inhibitors of Pyridoxal Kinase .… 63 5.1 Introduction…………………………………………………………… 63 5.2 Methods 5.2.1 Selection of Compounds for Inhibition Study ……………………. 63 5.2.2 Determination of Inhibition Constants …………………………….. 64 vii 5.2.3 Crystallization, Data Collection, and Structure Determination ……. 65 5.3 Results and Discussion 5.3.1 Inhibition studies ………………………………………………….... 69 5.3.2 Structure of PL kinase-Inhibitor Complex …………………………. 71 5.4 Conclusion …………………………………………………………… 76 References……………………………………………………………………………….. 77 VITA…………….……………………………………………………………………….. 93 viii List of Tables Page Table 1: Data collection and refinement statistics for the hPL kinase crystals................ 21 Table 2: Kinetic parameters of human PL kinase substrates (in µM)………………..…..23 Table 3: Crystal information, data collection and refinement parameters of D235A and D235N mutants............................................................................................. 41 Table 4: Kinetic parameters of hPL kinase wild type and D235A and D235N mutants ...42 Table 5: Refinement parameters for the human PL kinase structure with bound ATP and PLP. .............................................................................................................. 53 Table 6: Refinement parameters for the human PL kinase structure with bound theophylline ........................................................................................................ 68 Table 7: Kinetic parameters of human PL kinase inhibition by drugs ……..………..…..70 ix List of Figures Page Figure 1: Structures of the six B6 vitamers . ........................................................................... 1 Figure 2: Scheme for the salvage pathway of vitamin B 6 metabolism ….………………..…… 2 Figure 3: PL kinase catalyzed reaction . ................................................................................. 3 Figure 4: Homodimeric structure of ePL kinase with bound PL (grey) and ATP ( blue) . ......... 4 Figure 5: Reaction catalyzed by PNP oxidase ........................................................................ 5 Figure 6: Homodimeric structure of ePNP oxidase with bound FMN (red) and PLP (brown). 6 Figure 7: Synthesis of neurotransmitters by B 6 enzymes (colored red) ..................................... 9 Figure 8: Binding equilibria for human PL kinase substrates . ............................................... 22 Figure 9: Determination of KA. ........................................................................................... 24 Figure 10: Determination of KB. ......................................................................................... 24 Figure 11: Determination of αK A. ....................................................................................... 25 Figure 12: Determination of αK B. ....................................................................................... 25 Figure 13: Activity of human PL kinase in the presence of either Na + or K +. ......................... 26 Figure 14: Overall structure of hPL kinase. ( A) The monomeric structure with bound ATP (stick), MPD (stick), Na + (brown sphere), and Mg 2+ (magenta sphere) at the active site. α- Helices