University of South Florida Scholar Commons Graduate Theses and Dissertations Graduate School 2007 Examining the evolution of phosphagen kinases: A study of a dimeric arginine kinase from sea urchin (Strongylocentrotus purpuratus) eggs Brenda Christine Held University of South Florida Follow this and additional works at: http://scholarcommons.usf.edu/etd Part of the American Studies Commons Scholar Commons Citation Held, Brenda Christine, "Examining the evolution of phosphagen kinases: A study of a dimeric arginine kinase from sea urchin (Strongylocentrotus purpuratus) eggs" (2007). Graduate Theses and Dissertations. http://scholarcommons.usf.edu/etd/2208 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]. Examining the Evolution of Phosphagen Kinases: A Study of a Dimeric Arginine Kinase From Sea Urchin (Strongylocentrotus purpuratus ) Eggs by Brenda Christine Held A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemistry College of Arts and Sciences University of South Florida Major Professor: Steven H. Grossman, Ph.D. Li-June Ming, Ph.D. Randy Larsen, Ph.D. Brian Livingston, Ph.D. Date of Approval: April 2, 2007 Keywords: Enzyme characterization, purification, kinetics, echinoderm, sequence homolgy © Copyright 2007, Brenda Christine Held Dedication I would like to dedicate this to my family: Mom, Dad, and Toni. Without your love and support I would not have been able to do this. You are the ones who made me believe that I could be anything I wanted to be. Acknowledgements I would first like to thank Dr. Steven Grossman for all of his help and guidance throughout this process. You made me strive to better than I thought I could be. I cannot imagine going through graduate school without you as my major professor. If it had been anyone else, I would probably have quit somewhere along the way. I would also like to thank everyone in my family. You keep me grounded and don’t let me take myself too seriously. I always know that you are there for me and that you are always willing to come to my defense, even if you don’t know all the circumstances. Thanks also to all of my friends, both new and old. You kept me laughing and listened to me complain. You guys were one of the best and most unexpected blessings of this entire endeavor. I would also like to thank Dr. Livingston and Dr. Yoder for their help and guidance in the new and unfamiliar area of molecular biology. Thanks also to Dr. Ming and Dr. Larsen for agreeing to serve on my committee. Finally, and most importantly, I would like to thank God. He is the creator of all things and it is His masterpiece that makes the scientific research I do interesting and worthwhile. Note to Reader Note to Reader: The original of this document contains color that is necessary for understanding the data. The original dissertation is on file with the USF library in Tampa, Florida. Table of Contents List of Tables vi List of Figures vii List of Abbreviations x Abstract xii Chapter 1: Introduction 1 Background 1 Evolution 3 Sequences 7 Physical Characteristics and Kinetic Constants 14 Quaternary Structure 15 pH Optima 15 Temperature Stability 17 Isoelectric Point 18 Immunogenic Cross-reactivity 19 Synergism 20 Kinetic Constants 21 Mechanism 26 Active Site 30 i Structural Similarities 34 Conclusion 35 Chapter 2: Materials & Methods 36 General Procedures 36 Activity Assays 36 Reverse Direction 36 Forward Direction 36 SDS-PAGE 37 Protein Determination 38 Purification 38 Physical Characterization 41 Molecular Weight/Subunit Composition 41 Enzyme Stability 41 pH Stability 41 Effect of DTT 42 Effect of EDTA, MgCl 2, and ADP 42 Tris and Sodium Phosphate Buffer Stability 42 Thermal Inactivation 43 pH Optima 43 Isoelectric Point 44 Absorption Spectrum 44 Polyclonal Antibody/Western Blotting 44 ii Substrate Specificity 45 Hybridization 46 Kinetic Experiments 47 Reverse Kinetics (Formation of Arg and ATP) 47 Forward Kinetics (Formation of Arg~P and ADP) 47 Effect of Monovalent Anions 48 Nitrate Inhibition 48 Renaturation After Denaturation 48 Molecular Biology 49 RNA Isolation 49 cDNA Synthesis 49 DNA Concentration 50 DNA Gel Electrophoresis 50 Crude Mini-Prep 50 Eppendorf Perfect Prep 51 Restriction Digest 51 Design of Degenerate Primers 51 Insert Sequence PCR and Sequencing 52 Design of 5’ and 3’ Primers 53 5’ and 3’ PCR and Sequencing 54 5’-3’ Over-expression 55 Sequence Alignment/Homology 57 iii Chapter 3: Results 58 Purification 58 Physical Characterization 58 Molecular Weight/Subunit Composition 58 Enzyme Stability 64 pH Stability 64 Effect of DTT 64 Effect of EDTA, MgCl 2, and ADP 64 Tris and Sodium Phosphate Buffer Stability 64 Thermal Inactivation 69 pH Optima 69 Isoelectric Point 69 Absorption Spectrum 69 Polyclonal Antibody/Western Blotting 74 Substrate Specificity 74 Hybridization 74 Kinetic Experiments 77 Reverse Kinetics (Formation of Arg and ATP) 77 Forward Kinetics (Formation of Arg~P and ADP) 77 The Effect of Monovalent Anions 78 Nitrate Inhibition 88 Renaturation After Denaturation 88 iv Molecular Biology 88 Design of Degenerate Primers 88 Insert Sequence PCR and Sequencing 91 Design of 5’ and 3’ Primers 91 5’ and 3’ PCR and Sequencing 91 5’-3’ Over-expression 92 Sequence Alignment/Homology 93 Chapter 4: Discussion 96 Purification and Quaternary Structure 96 pH Optima and Isoelectric Point 98 Temperature Stability 101 Immunogenic Cross-reactivity 102 Substrate Specificity 103 Hybridization 104 Synergism 105 Kinetic Characteristics 107 Monovalent Anions 109 Structural Similarities 111 Sequence Homology and Evolutionary Relationships 112 Conclusions 116 Further Research 118 References 119 About the Author End Page v List of Tables Table 1: pH optima for various AK and CK enzymes 16 Table 2: Temperature stability of various AK and CK enzymes 18 Table 3: Isoelectric points of various AK and CK enzymes 19 Table 4: Kinetic constants for various AK and CK enzymes in the forward 24 direction Table 5: Kinetic constants for various AK and CK enzymes in the reverse 25 direction Table 6: Catalytic efficiency for various CK and AK enzymes 26 Table 7: Purification Table for Sea Urchin Egg Arginine Kinase 61 Table 8: Kinetic Constants for both the Forward and Reverse Reactions 85 Table 9: Percent Inhibition of Various Monovalent Anions 87 Table 10: Molecular Weight/Subunit Composition of Some Phosphagen Kinases 98 Table 11: Synergism in Phosphagen Kinases 107 vi List of Figures Figure 1: Possible Evolutionary Scheme for the phosphagen kinases 7 Figure 2: Alignment of Dimeric AK against Representative CK enzymes 10 Figure 3: Alignment of Dimeric AK against Representative AK enzymes 11-12 Figure 4: Arginine and ADP Binding Sites for Arginine Kinase From Limulus 14 Figure 5: Schematic of the Rapid Random Equilibrium Reaction of Arginine and 28 Creatine Kinases Figure 6: Structure of Arginine Kinase Transition State Analog From Limulus 31 Figure 7: AK Superimposed onto CK 34 Figure 8: Enzymatic Assays for both the forward and reverse direction 37 Figure 9: Purification of Arginine Kinase from Sea Urchin Eggs Flow Chart 40 Figure 10: Micro titer plate assay used in the reverse direction pH optima 44 determination Figure 11: Electrophoretic Colorimetric Enzymatic Assay 46 Figure 12: ACA Column Elution Profile 59 Figure 13: DEAE Sepharose Column Elution Profile 60 Figure 14: Purification SDS-PAGE Gel 62 Figure 15: Molecular Weight Determination of Sea Urchin Egg AK 63 Figure 16: pH Stability 65 vii Figure 17: pH Stability Day 3, Day 6, & Day 12 66 Figure 18: Effect of DTT, EDTA, MgCl 2, and ADP 67 Figure 19: Stability in Tris/HCl and Sodium Phosphate Buffers 68 Figure 20: Temperature Stability 70 Figure 21: pH Optima in the Forward and Reverse Direction 71 Figure 22: Isoelectric Focusing 72 Figure 23: Absorption Spectrum for purified sea urchin egg AK 73 Figure 24: Western Blot Using Polyclonal Antibody Against Sea Urchin 75 Egg AK Figure 25: Hybridization of Sea Urchin Egg AK with Sea Cucumber Muscle AK 76 Figure 26: Effect of Phosphoarginine 79 Figure 27: Effect of ADP 80 Figure 28: Secondary Plot for the Reverse Direction (Formation of Arg and 81 ATP) Figure 29: Effect of Arginine 82 Figure 30: Effect of ATP 83 Figure 31: Secondary Plot for the Forward Reaction (Formation of Arg~P 84 and ADP) Figure 32: Monovalent Anion Time Courses 86 Figure 33: Varying Nitrate Concentrations Time Courses 89 Figure 34: Percent Inhibition of Various Nitrate Concentrations 90 Figure 35: Over-expression of 5’-3’ insert over 3 hours 94 Figure 36: Purification of Over-expression of 5’-3’ Insert 95 viii Figure 37: Binding Site for Arginine and Creatine in Arginine and Creatine 104 Kinases Figure 38: Sequence Alignment of Dimeric CK, Dimeric AK, and Monomeric 112 AK Figure 39: Phylogenetic Tree of the Relationship of Vertebrates and 114 Invertebrates Figure 40: Phylogenetic Tree of a Variety of Phosphagen Kinases From Both 116 Vertebrates and Invertebrates ix List of Abbreviations AK: Arginine Kinase CK: Creatine Kinase LK: Lombricine Kinase GK: Glycocyamine Kinase TK: Taurocyamine Kinase HK: Hypotaurocyamine Kinase kD: kilo Daltons pI: Isoelectric Point ADP: Adenosine 5’ diphosphate ATP: Adenosine 5’ triphosphate DEAE: Diethylaminoethyl DTT: Dithiothreitol EDTA: Ethylenediaminetetraacetic acid NADP +: Nicotinamide adenine dinucleotide phosphate, oxidized form NADPH: Nicotinamide adenine dinucleotide phosphate, reduced form PAGE: Polyacrylamide gel electrophoresis SDS: Sodium dodecyl sulfate Tris: Tris(hydroxymethyl)aminomethane x DNA: Deoxyribonucleic acid RNA: Ribonucleic acid cDNA: Complementary DNA DEPC: Diethylpyrocarbonate xi Examining the Evolution of Phosphagen Kinases: A Study of a Dimeric Arginine Kinase From Sea Urchin (Strongylocentrotus purpuratus ) Eggs Brenda Held ABSTRACT The phosphagen kinases are a family of enzymes that catalyze the reversible phosphorylation of specific phosphagens using ATP as the phosphate donor.