University of South Florida Scholar Commons Graduate Theses and Dissertations Graduate School 2009 Investigation into the rate-determining step of mammalian heme biosynthesis: Molecular recognition and catalysis in 5-aminolevulinate synthase Thomas Lendrihas University of South Florida Follow this and additional works at: http://scholarcommons.usf.edu/etd Part of the American Studies Commons Scholar Commons Citation Lendrihas, Thomas, "Investigation into the rate-determining step of mammalian heme biosynthesis: Molecular recognition and catalysis in 5-aminolevulinate synthase" (2009). Graduate Theses and Dissertations. http://scholarcommons.usf.edu/etd/2059 This Dissertation is brought to you for free and open access by the Graduate School at Scholar Commons. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact [email protected]. Investigation into the Rate-Determining Step of Mammalian Heme Biosynthesis: Molecular Recognition and Catalysis in 5-Aminolevulinate Synthase by Thomas Lendrihas A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Molecular Medicine College of Medicine University of South Florida Major Professor: Gloria C. Ferreira, Ph.D. Samuel I. Beale, Ph.D R. Kennedy Keller, Ph.D. Randy W. Larsen, Ph.D. Gene C. Ness, Ph.D. Larry P. Solomonson, Ph.D. Date of Approval: June 30, 2009 Keywords: X-linked sideroblastic anemia, α-oxoamine synthase, transient kinetics, pyridoxal 5'-phosphate, porphyria, photodynamic therapy © Copyright 2009, Thomas Lendrihas Acknowledgements I wish to express my gratitude to the members of my committee, Dr. R. Kennedy Keller, Dr. Randy W. Larsen, Dr. Gene C. Ness, and Dr. Larry P. Solomonson for their consistent guidance, understanding and support throughout the course of my graduate work. Most of all, to Dr. Gloria C. Ferreira, I am deeply appreciative for allowing me the privilege of working with her side-by-side. Her remarkable guidance as both a scientific mentor and cherished friend will never be forgotten. I am grateful to all the professors and colleagues in the Department of Molecular Medicine, for their intellectual and personal contributions. To Dr. Gregory A. Hunter and Dr. Tracy D. Turbeville, I am indebted for both their scientific and emotional counsel. I wish to express my appreciation to Ms. Kathy Zahn and Ms. Maxine Roth at the Office of Research and Graduate Affairs for their continuous administrative assistance. Additionally, I would like to specifically thank Ms. Helen Chen-Duncan for her unwavering support and caring as both a colleague and treasured friend. I am forever grateful to my friends: Zena Y. Davis, Julia B. Huddleston, John K. Knowles, Mitchell M. McNelly, Laura Jackson Roberts, Louis J. Smith and Thomas F. Zarella, for their enduring encouragement and love. Finally, I wish to acknowledge my family, without whom, this journey would not have been possible. Table of Contents List of Tables iii List of Figures iv List of Abbreviations vi List of Schemes ix Abstract x Chapter One 1 INTRODUCTION: The central function of heme: biogenesis, chemistry and health 1 Enzymes in the heme biosynthesis pathway 2 Aminolevulinate synthase 2 Porphobilinogen synthase 11 Porphobilinogen deaminase 13 Uroporphyrinogen III synthase 15 Uroporphyrinogen decarboxylase 19 Coproporphyrinogen oxidase 21 Protoporphyrinogen oxidase 24 Ferrochelatase 26 Enzymes in the heme degradation pathway 31 Heme oxygenase 31 Biliverdin reductase 34 Content of the dissertation 36 References 36 Chapter Two 51 SERINE-254 ENHANCES AN INDUCED FIT MECHANISM IN MURINE i 5-AMINOLEVULINATE SYNTHASE 51 Abstract 51 Introduction 53 Materials 59 Methods 59 Results 65 Discussion 74 Acknowledgements 79 References 79 Chapter Three 82 ARG-85 AND THR-430 IN MURINE 5-AMINOLEVULINATE SYNTHASE COORDINATE ACYL-COA-BINDING AND CONTRIBUTE TO SUBSTRATE SPECIFICITY 82 Abstract 82 Introduction 84 Materials 88 Methods 88 Results 93 Discussion 106 Acknowledgements 114 References 114 Chapter Four 117 HYPERACTIVE ENZYME VARIANTS ENGINEERED BY SYNTHETICALLY SHUFFLING A LOOP MOTIF IN MURINE 5-AMINOLEVULINATE SYNTHASE 117 Abstract 117 Introduction 119 Materials 123 Methods 123 Results 132 Discussion 150 Acknowledgements 156 References 156 Chapter Five 159 SUMMARY AND CONCLUSION 159 References 167 About the Author End Page ii List of Tables Table 2.1. Summary of steady-state kinetic parameters. 66 Table 2.2. Gibb’s free energy associated with the S254 variant-catalyzed reactions. 78 Table 3.1. Comparison of steady-state kinetic constants for wild-type ALAS, R85K, R85L, and R85L/T430V with CoA derivatives as substrates. 95 Table 3.2. Rates of quinonoid intermediate formation and decay under single- turnover conditions. 103 Table 4.1. Designed mutations for incorporation at indicated positions within the ALAS active site loop. 124 Table 4.2. Amino acids substitutions in active site lid variants. 139 Table 4.3. Kinetic parameters for the reactions of hyperactive ALAS enzymes. 141 Table 4.4. Thermodynamic activation parameters of wild-type ALAS and the SS2 variant. 149 iii List of Figures Figure 1.1. Enzymes and intermediates of the heme biosynthetic pathway. 4 Figure 1.2. The X-ray crystal structure of porphobilinogen deaminase from Homo sapiens. 14 Figure 1.3. The three-dimensional structure of human uroporphyrinogen III synthase. 16 Figure 1.4. The X-ray crystal structures of coproporphyrinogen III oxidase. 22 Figure 1.5. The three-dimensional structure of ferrochelatase from Homo sapiens. 28 Figure 1.6. Enzymes in the heme degradation pathway. 32 Figure 2.1. Structural models for murine erythroid ALAS based on the R. capsulatus crystal structures. 57 Figure 2.2. Multiple sequence alignment of phylogenetically diverse members of the α-oxoamine synthase family in the region of murine eALAS serine-254. 58 Figure 2.3. Circular dichroism and fluorescence emission spectra of ALAS and the S254 variants. 67 Figure 2.4. Reaction of the S254 variants (60 µM) with increasing concentrations of glycine. 69 Figure 2.5. Reaction of wild-type ALAS and the S254 variants (5 µM) with ALA. 70 Figure 2.6. Reaction of wild-type ALAS- and S254 variant-glycine complexes with succinyl-CoA under single turnover conditions. 72 iv Figure 2.7. Kinetic mechanisms of the S254 variant enzymes. 78 Figure 3.1 The acyl-CoA-binding cleft in R. capsulatus ALAS. 87 Figure 3.2. Comparison of normalized specificity constants for murine eALAS variants with different CoA substrates. 98 Figure 3.3. Visible circular dichroism spectra of wild-type ALAS and the R85 and R85/T430 variants. 99 Figure 3.4. Reaction of wild-type ALAS, R85K, R85L and R85L/T430V (5 µM) with ALA. 101 Figure 3.5. Reaction of wild-type ALAS- and R85K-glycine complexes with different CoA derivatives under single turnover conditions. 104 Figure 4.1. The position of the active site loop in the R. capsulatus ALAS crystal structure. 122 Figure 4.2. The generation and screening of the library. 126 Figure 4.3. Differential fluorescence of ALAS variant isolates streaked on expression agar. 128 Figure 4.4. Multiple alignment of the amino acid sequences of the ALAS loop region. 134 Figure 4.5. The single turnover reactions of isolated hyperactive ALAS variants. 141 Figure 4.6. The SS2 variant-catalyzed reaction. 147 Figure 4.7. The thermal dependence of the SS2-variant catalyzed reaction. 148 Figure 4.8. The simulated kinetic mechanism of the SS2 variant-catalyzed reaction. 149 v List of Abbreviations A-site Acetyl-site AAT Aspartate aminotransferase AIP Acute intermittent porphyria ALA 5-Aminolevulinate ALAD 5-Aminolevulinate dehydratase ALAS 5-Aminolevulinate synthase ALAS1 5-Aminolevuinate synthase (Non-specific isoform) ALAS2 5-Aminolevulinate synthase (Erythroid-specific isoform) AON 8-Amino-7-oxononanoate AONS 8-Amino-7-oxononanoate synthase Bach-1 Basic leucine transcription factor 1 BVR Biliverdin reductase CD Circular dichroism CO Carbon monoxide CO2 Carbon dioxide CoA Coenzyme-A CEP Congenital erythropoietic porphyria CPK Corey, Pauling and Koulton vi CPO Coproporphyrinogen oxidase DEAE Diethylaminoethyl EPP Erythropoietic protoporphyria FAD Flavin adenine dinucleotide FC Ferrochelatase GATA1 Globin transcription factor 1 HCP Hereditary coproporphyria HEP Hepatoerythropoietic porphyria HEPES (N-[2-Hydroxyethyl] piperazine-N’-[2-ethane sulfonic acid]) HMB Hydroxymethylbilane HO Heme oxygenase HRM Heme regulatory motif INH 4-Bromo-3-(5'-carboxy-4'-chloro-2'-fluoro-phenyl)-1-methyl-5- trifluoromethyl-pyrazol IRE Iron response element IRP IRE-binding protein KBL 2-Amino-3-ketobutyrate-CoA ligase meALAS Murine erythroid ALAS mno montalcino (zebrafish variant displaying defective PPO activity) MOPS 4-Morpholinepropanesulfonic acid NAD+ -Nicotinamide adenine dinucleotide NADPH -Nicotinamide adenine dinucleotide phosphate O2 Diatomic oxygen vii PBG Porphobilinogen PBGD Porphobilinogen deaminase PBGS Porphobilinogen synthase PCT Porphyria cutanea tarda P-site Propionyl-site PDB Protein data bank PLP Pyridoxal 5’-phosphate PPO Protoporphyrinogen oxidase RMSD Root mean square deviation SAM S-adenosyl-L-methionine SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SPT Serine palmitoyl transferase SS2 Synthetically shuffled variant #2 UROD Uroporphyrinogen decarboxylase UROS Uroporphyrinogen synthase VP Variegate porphyria XLSA X-linked sideroblastic anemia viii List of Schemes Scheme 1.1. The chemical mechanism of