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MECHANISTIC STUDIES on PHOSPHOGLUCOMUTASE By MECHANISTIC STUDIES ON PHOSPHOGLUCOMUTASE By MICHAEL DAVID PERCTVAL B.Sc. (Hons), The University of Otago, 1982 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Chemistry) We accept this thesis as conforming toihe required standard THE UNIVERSITY OF BRITISH COLUMBIA September 1988 © Michael David Percival, 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Chemistry The University of British Columbia Vancouver, Canada Date 12 October, 1988 DE-6 (2/88) ii ABSTRACT. The mechanism of rabbit skeletal muscle phosphoglucomutase (EC.2.7.5.1) has been investigated using fluorinated and deoxygenated substrate analogues. Each of the analogues in which the non-acceptor hydroxyls are replaced by fluorine or hydrogen are substrates of the enzyme. The kinetic constants of these substrates are reported. The rate of the mutase reaction of each substrate analogue in the presence of glucose 1,6-diphosphate is the same as that of the half reaction involving production of the fluorinated and deoxygenated glucose 1,6-diphosphate species. The exceptions are 3-fluoro- and 3-deoxy-glucose 1-phosphate, in which cases the rates of the half reactions are 8 times that of the overall mutase reaction. The Km of 3-fluoro-glucose 1,6-diphosphate is approximately 90 times that of glucose 1,6-diphosphate and the other deoxy and fluoro analogues. The inhibition of phosphoglucomutase by fluorinated and deoxygenated substrate analogues has been investigated. The synthesis of a series of novel disubstituted inhibitors (based on glucose 1-phosphate) in which the C-6 hydroxyl is replaced by fluorine and a sugar ring hydroxyl is replaced by either hydrogen or fluorine is described. The inhibition constants show that the hydroxyl distal to the acceptor hydroxyl is most important in the formation of a strong enzyme-inhibitor complex. The synthesis is described of three phosphorofluoridate analogues of glucose phosphate substrates. These analogues were found to only weakly inhibit phosphoglucomutase. No evidence of any phosphoryl transfer between the phosphoenzyme and the phosphorofluoridate analogues could be detected. Thus phosphoglucomutase has a strict requirement for a doubly negatively charged substrate phosphate group. The interaction of phosphoglucomutase with fluorinated substrates and inhibitors has been investigated by l^F-nmr. Large downfield changes in the chemical shifts of the inhibitors 6-fluoro-glucose 1-phosphate and a-glucosyl fluoride 6-phosphate were found to accompany binding to the phosphoenzyme. The effects of the binding of activating and non-activating metal ions on these spectra were investigated. The different effects observed may be directly related to the chemical basis for the metal induced activation of the enzyme. ^F-Nmr data consistent with i i i a 102 to 103 fold increase in the tenacity with which phosphoglucomutase binds substrates and inhibitors in the presence of Li+ were observed in the spectra of the phosphoenzyme with difluorinated glucose 1-phosphate inhibitors. Two enzyme bound species were detected in the l^F-nrnr spectra of the complexes formed by reaction of the Cd^+.phosphoenzyme with 2- and 3-fluoro-glucose phosphates. These species are tentatively assigned as the fluoro-glucose 1,6- diphosphate species bound in two different modes to the dephosphoenzyme. Only one bound species was observed in the case of 4-fluoro-glucose phosphates. The environment of each substrate glucose hydroxyl in the active site was probed using l^p-rimr and the fluorinated glucose phosphate substrates. Data inconsistent with a minimal motion type of mechanism (WJ. Ray, A.S. Mildvan & J.W. Long, Biochemistry 1973,12, 3124) were obtained. The results of the nmr and kinetic studies are consistent with an exchange type of mechanism in which the C-3 hydroxyl plays an important role in the reorientation of the glucose 1,6-diphosphate. The data also suggest that there are two distinct glucose binding sites, one for each substrate and glucose 1,6-diphosphate bound in the same mode. i v TABLE OF CONTENTS. Abstract ii List of Tables vii List of Figures viii List of Abbreviations xi Glossary of Enzymic Terms xiii Acknowledgements xiv I. Background 1 1.1. Enzymes-A General Background and Perspective 1 1.2. Phosphoglucomutase-A Brief Introduction to the Enzyme 3 1.2.1. History and Biological Significance 3 1.2.2. The Mechanism of the Reaction 4 1.2.3. Metal Ion Effects 9 1.2.4. Other Phosphomutases 10 1.2.5. Aims of this Study 12 n. Kinetic Studies on the Utilization of Fluorinated and Deoxygenated Substrate Analogues 14 II.A. Introduction 14 II.A.l. The Use of Enzyme-Substrate Binding Energy 14 II. A.2. The Source of Enzyme-Substrate Binding Energy and its Measurement 16 II.A.3. The Effects of Ligand Modification 19 II.A.4. The Energetics of Hydrogen Bond Formation 24 II.A.5. Summary and Overview 25 II.B. Results and Discussion 27 II.B.1. The Synthesis of Fluorinated and Deoxygenated Analogues of Glucose 1- Phosphate 27 II.B. 1.1. 2,6-Dideoxy-2,6-difluoro-a-D-glucopyranosyl phosphate .... 27 II.B. 1.2. 3,6-Dideoxy-3,6-difluoro-a-D-glucopyranosyl phosphate .... 28 II.B. 1.3. 3,6-Dideoxy-6-fluoro-a-D-ribohexopyranosyl phosphate 31 II.B. 1.4. 4,6-Dideoxy-4,6-difluoro-a-D-glucopyranosyl phosphate .... 33 II.B. 1.5. 4,6-Dideoxy-6-fluoro-a-D-xylohexopyranosyl phosphate .... 35 II.B. 1.6. 2-Deoxy-a-D-arabinohexopyranosyl phosphate 37 II.B.2. Analysis of the Solution Conformations of Glucose 1-Phosphate Analogues 40 II.B.3. Interaction of Fluoro and Deoxy-Substrates and Inhibitors with Phosphoglucomutase 43 II.B.3.1. Methods of the Kinetic Parameter Analysis 43 II.B.3.2. Substrate Activity of Fluoro and Deoxy-Analogues 44 II.B.3.3. Determination of Substrate Kinetic Parameters 52 II.B.3.4. Determination of Inhibitor Kinetic Parameters 59 II.B.3.5. Spectral Studies on Enzyme-Ligand Complexes 66 II.B.3.6. Evaluation of Equilibrium Constants of Fluoro-Substrates .... 69 II.B.4. Summary and Implications for the Mechanism 70 II.C. Experimental Procedures 74 II.C.l. General Synthetic Methods and Purification of Reagents 74 V II.C.2. Synthetic Methods 77 II.C.3. Biological Methods 92 HI. Charge State Analogues and Potential Covalent Inhibitors 95 III.A. Introduction 95 III.A.l. Substrate Ionization States 95 III.A.2. Phosphite Esters as Phosphate Analogues 98 III.A.3. Phosphorofluoridate Esters as Phosphate Analogues 100 III.A.4. Cyclic-Phosphate Esters as Phosphate Analogues 105 III.A.5. Sulfate Esters as Phosphate Analogues 106 III.A.6. Enzyme-Phosphate Electrostatic Interactions in Phosphoglucomutase 107 III.A.7. Summary and Overview 108 III.B. Results and Discussion 109 III.B.l. Synthesis of Phosphorofluoridate Analogues 109 III.B.2. Synthesis of Cyclic-Phosphate Analogues 112 III.B.3. Biological Activity of Phosphorofluoridate and Cyclic-Phosphate Analogues 113 III. C. Experimental Procedures 121 III.C.l. General Methods and Purification of Reagents 121 III. C.2. Synthetic Methods 122 IV. NMR Studies of Enzyme-Ligand Complexes 126 IV. A. Introduction 126 IV. A.l. 19F-NMR in Macrolecular Systems 126 IV.A.2. 19F-NMR Chemical Shifts in Macromolecular Systems 128 IV.A.3. Relaxation in Macromolecular Systems 130 IV.A.4. Nuclear Overhauser Effects in Macromolecular Systems 132 IV.A.5. Summary and Overview 133 IV.B. Results and Discussion 134 IV.B.1. Synthesis of Fluorinated and Deoxygenated Analogues of Glucose 6- Phosphate 134 IV.B.2. Investigation of Enzyme-Ligand Complexes by ^9F-NMR 137 IV.B.2.1. Complexes of 6-Fluoro-Glucose 1-Phosphate and oc-Glucosyl Fluoride 6-Phosphate 137 IV.B.2.2. Effect of Binding of Activating Metal Ions 152 IV.B.2.3. Ternary Enzyme-Cd2+-Fluoro-Substrate Complexes 161 IV.B.2.4. Complexes of Difluorinated Inhibitors 170 IV.B.2.5. Ternary Phosphoenzyme-Li+-Ruoro-Substrate Complexes 177 IV.B.3. Summary and Implications for the Mechanism 194 IV.B.4. 19F.1H. Nuclear Overhauser Effects 196 IV.B.4.1. Investigation of a Ligand-Enzyme iH-f^F} NOE 198 IV.B.4.2. Investigation of a Ligand-Enzyme 19F-{!H} NOE 201 IV.C. Experimental Procedures 205 IV.C.l. General 205 IV.C.2. Synthetic Methods 205 IV.C.3. Enzyme Isolation and Purification 207 IV.C.4. Demctallation of NMR Solutions and Additives 211 IV.C.5. Demctallation of Phosphoenzyme 212 IV.C.6. Acquisition of 19F-NMR Spectra 214 IV.C.7. Deuteration of Phosphoenzyme and Reagents for ^H-NMR 215 IV.C.8. Acquisition of ifl-NMR Spectra 215 V. Summary 217 vi Appendix 221 References 229 vii LIST OF TABLES. Table II. 1. Comparison of the sizes of some funtional groups 20 Table 1X2. ^H, l^F and 31p-nmr chemical shifts of substituted a-D-glucopyranosyl phosphates 41 Table 1X3. lH-lH coupling constants of substituted a-D-glucopyranosyl phosphates 41 Table 1X4. ^H-^lp and ^H-l^F coupling constants of substituted a-D-glucopyranosyl phosphates 42 Table 1X5. Kinetic constants of fluorinated and deoxygenated substrate analogues of glucose 1-phosphate with phosphoglucomutase 54 Table 1X6.
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