X-Ray Crystallographic Studies of the Metal Binding Sites on Recombinant Porcine Fructose-1,6-Bisphosphatase Jun-Yong Choe Iowa State University
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Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 2001 X-ray crystallographic studies of the metal binding sites on recombinant porcine fructose-1,6-bisphosphatase Jun-yong Choe Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Biophysics Commons Recommended Citation Choe, Jun-yong, "X-ray crystallographic studies of the metal binding sites on recombinant porcine fructose-1,6-bisphosphatase " (2001). Retrospective Theses and Dissertations. 483. https://lib.dr.iastate.edu/rtd/483 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. 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This microform edition is protected against unauthorized copying under Title 17, United States Code. Bell & Howell Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 ii Graduate College Iowa State University This is to certify that the doctor dissertation of Jun-yong Choe has met the dissertation requirements of Iowa State University Signature was redacted for privacy. Major P ofess Signature was redacted for privacy. For the Major Program Signature was redacted for privacy. For the Graduate College iii TABLE OF CONTENTS ABBREVIATIONS ABSTRACT CHAPTER 1. GENERAL INTRODUCTION I Background of FBPase 1 Properties of FBPase 3 Questions Regarding the Allosteric Regulation by AMP and the Role of Loop 52-72 16 The Role of Loop 52-72 and Allosteric Transition 19 Monovalent Metal Sites 21 Catalytic Mechanism of FBPase 24 Dissertation Organization 28 References 30 CHAPTER 2. ROLE OF DYNAMIC LOOP IN CATION ACTIVATION AND ALLOSTERIC REGULATION OF RECOMBINANT POCBME FRUCTOSE 1,6-BISPHOSPHATASE 38 Abstract 38 Introduction 39 Experimental Procedures 41 Results 44 Discussion 59 References 66 CHAPTER 3. CRYSTAL STRUCTURES OF FRUCTOSE 1,6-BISPHOSPHATASE: MECHANISM OF CATALYSIS AND ALLOSTERIC INHIBITION REVEALED IN PRODUCT COMPLEXES 71 Abstract 71 Introduction 72 Experimental Procedures 75 Results 78 Discussion 89 References 96 iv CHAPTER 4. CRYSTALLOGRAPHIC STUDIES FOR THE MONOVALENT CATION SITES FOR THALLIUM ON FRUCTOSE-1,6 -BISPHOSPHATASE 101 Abstract 101 Introduction 102 Experimental Procedures 104 Results 107 Discussion 117 References 124 CHAPTER 5. METAPHOSPHATE IS CAPTURED IN CRYSTAL STRUCTURES OF FRUCTOSE 1,6-BISPHOSPHATASE 128 Abstract 128 Report 129 References and Notes 138 CHAPTER 6. CONCLUSION 141 Summary 141 APPENDIX 1. DIRECTED MUTATIONS IN THE POORLY DEFINED REGION OF PORCINE LIVER FRUCTOSE-1,6-BISPHOSPHATASE SIGNIFICANTLY AFFECT CATALYSIS AND THE MECHANISM OF AMP INHIBITION 144 APPENDIX 2. TRYPTOPHAN FLUORESCENCE REVEALS THE CONFORMATIONAL STATE OF A DYNAMIC LOOP IN RECOMBINANT PORCINE FRUCTOSE-1,6-BISPHOSPHATASE 151 APPENDIX 3. MUTATIONS IN THE HINGE OF DYNAMIC LOOP BROADLY INFLUENCE FUNCTIONAL PROPERTIES OF FRUCTOSE-1,6- BISPHOSPHATASE 159 ACKNOWLEDGMENTS 167 V ABBREVIATIONS AMP: adenosine monophosphate FBPase: fructose-1,6-bisphosphatase Pi: phosphate F6P: fructose 6-phosphate FI6P2: fructose 1,6-bisphosphate F26P%: fructose 2,6-bisphosphate 2,5-AnG-l,6-P2-. 2,5-anhydroglucitol 1,6-bisphosphate To indicate amino acids in protein sequences or names of the mutant enzymes, one letter or three letters abbreviation was used. vi ABSTRACT Fructose 1,6-bisphospbatase (D-ftuctose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11, FBPase; Fructose 1,6-bisphosphate + H2O o Fructose 6-phosphate + Phosphate) is a key regulatory enzyme in gluconeogenesis. It is a homotetramer with subunit molecular mass of 37 kDa. There are two different quaternary conformations of FBPase: the R-state (the active conformation) and the T-state (the inactive conformation). FBPase is inhibited allosterically by AMP. FBPase requires divalent metal ions (Mg2r, Mn2", Zn2", or Co2") to hydrolyze fructose 1,6-bisphosphate. Some monovalent cations (K~, Rtf, Tl™ or NH3*) maximize the enzyme activity, whereas lithium ions inhibit the enzyme. Li1- inhibition of FBPase is similar to its effect on inositol monophosphatase, and, indeed FBPase and myo-inositol monophosphatase share a common polypeptide fold. FBPase is a target for the development of drugs in treating non-insulin dependent diabetes. A dynamic loop 52-72, revealed in a crystal structure of a product complex, and interacts with the active site of the enzyme. Loop 52-72 plays an important role in the allosteric mechanism, existing in two different conformations. A new divalent metal site and four monovalent metal sites were discovered. All of metal sites could be important in vivo. FBPase might select a combination of metals for activity in vivo. AMP competes with divalent metals and displaces loop 52-72 from the active site. Two distinct conformations for the 1-OH group of fructose 6-phosphate correspond to non-productive and productive states of ligation. Direct evidence for the existence of metaphosphate is presented in the active vii site of FBPase at near atomic resolution. The presence of the metaphosphate implies a dissociative mechanism in FBPase catalysis: The cleavage of the ester bond between atoms 0-1 and P of the fructose ^-diphosphate precedes the nucleophilic attack of water. 1 CHAPTER 1. GENERAL INTRODUCTION Background of FBPase Fructose 1,6-bisphosphatase (D-fructose- 1,6-bisphosphate 1 -phosphohydrolase, EC 3.1.3.11, FBPase; Fructose 1,6-bisphosphate + H2O o Fructose 6-phosphate + Phosphate) is a key regulatory enzyme in gluconeogenesis (1-7; Figure 1). Gluconeogenesis, the synthesis of glucose from non-carbohydrate precursors (8), is the reverse process of glycolysis, consumes energy and provides glucose to fasted animals. Gluconeogenesis in plants is involved in conversion of light energy into carbohydrate metabolites. Glucose or its derivatives are required by all cells for synthesis of glycolipids, glycoproteins, and other structural polysaccharides. Furthermore, it is the only energy source for mammalian erythrocytes and avian retina, and the main energy source for brain tissue. The glucose level in blood is crucial for mammalian. A short period of hypoglycemia may lead to irreversible damage to the brain (4). There are three regulatory cycles for the control of glucose levels in glycolysis and gluconeogenesis; glucose/glucose 6-phosphate cycle, fructose 6-phosphate/ftuctose 1,6-bisphosphate cycle, and pyruvate/phosphoenolpyruvate cycle. FBPase was discovered by Gomori in 1943 (9). FBPase is mainly present in gluconeogenic tissues like liver and kidney, but is also found in mammalian brain and skeletal muscle (3-5). FBPase has been isolated from various sources; porcine kidney (10), porcine liver (11), rabbit liver, kidney and muscle (12), mouse liver, rat liver (13) 2 Glucose Glycogen Triacylglycerols glucose-6- glycogen glycogen triacylglycerol "hormone- phosphatase kinase synthase phosphorylase synthesis sensitive" triacylglycerol lipase Glucose-6-phosphate Fatty acids fructose-1,6- phosphofructokinase bisphosphatase gluconeo fatty aad p oxidation genesis glycolysis synthesis pathway Phosphoenol- NAPDH J NADH + FADH, pyruvate NADPH pyruvate kinase Pyruvate pyruvate lactate dehydro dehydrogenase genase pyruvate carooxylase phosphoenol- pyruvate cartioxykinase Ketone oodies t Ketogenic amino acids Glucogenic • Oxaioacetate amino acids -i NADH* FADH 2 Oxidative phosphorylation ; ATP Figure 1. Major energy metabolism pathways (7). 3 bovine