Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1990 The 5Å esolutr ion crystal structure of adenylosuccinate synthetase from Escherichia coli Michael Alan Serra Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Bacteriology Commons, Biochemistry, Biophysics, and Structural Biology Commons, and the Cellular and Molecular Physiology Commons Recommended Citation Serra, Michael Alan, "The 5Å er solution crystal structure of adenylosuccinate synthetase from Escherichia coli " (1990). Retrospective Theses and Dissertations. 11220. https://lib.dr.iastate.edu/rtd/11220 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. 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Ann Arbor, MI 48106 The 5Â resolution crystal structure of adenylosuccinate synthetase from Escherichia coli by Michael Alan Serra A Dissertation Submitted to the Graduate Faculty in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Department: Biochemistry and Biophysics Major; Molecular, Cellular, and Developmental Biology Approved: Signature was redacted for privacy. In Charge of Major Work Signature was redacted for privacy. For the Majo^VDepartment Signature was redacted for privacy. For the Graduate College Iowa State University Ames, Iowa 1990 i i TABLE OF CONTENTS Page DEFINITIONS iv ABBREVIATIONS v INTRODUCTION 1 Properties of the Synthetase from Escherichia coli 3 Mechanism 4 Role in Disease 6 Cancer 6 Hyperuricemia and gout 7 Malaria 8 Inhibitors of Therapeutic Importance 9 Long Range Goals 11 Present Study 12 CRYSTALLIZATION AND CHARACTERIZATION OF THREE CRYSTAL FORMS 13 Enzyme Preparation 13 Growth of the P2i and the P2i2j2]^ Crystal Forms 13 Crystalline Enzyme-Ligand Complexes; Soaking Experiments 16 Crystalline Enzyme-Ligand Complexes: Growth of the P3i21 (P3221) Crystal Form 16 Space Group Determination 18 Determination of the P2i crystal form 18 Determination of the ?2i2i2i crystal form 20 Determination of the P3i21 (P3221) crystal form 21 Results and Discussion 28 i i i STRUCTURE DETERMINATION OF THE P2i CRYSTAL FORM TO 5.0À RESOLUTION 32 Introduction 32 Phase Problem 36 Isomorphous replacement 37 Anomalous scattering 39 Materials and Methods 43 Crystallization 43 Crystal manipulation 44 Data collection 46 Data processing 49 RESULTS AND DISCUSSION 72 Stabilization Buffer 72 Heavy Atom Derivatives 74 Patterson Map 78 Rotation Function 80 Electron Density Map 87 APPENDIX: STRUCTURE OF l-(p-NITROBENZYLIDINEAMINO) GUANIDINIUM CHLORIDE 102 Abstract 102 Introduction 102 Experimental 104 Discussion 105 REFERENCES 112 ACKNOWLEDGEMENT 118 iv DEFINITIONS x,y,z fractional coordinates; for atomic positions a,b,c unit cell edge vectors parallel to the X, Y, and Z axes of a right handed coordinate system a*,b*,c* reciprocal unit cell vectors associated with the X, Y, and Z axes of a right handed coordinate system a,b,c unit cell edges in direct space a*,b*,c* unit cell edges in reciprocal space r direct space vector; r = ax + by + cz h reciprocal space vector; h = ha* + kb* + Ic* F(h) structure factor for the hth reciprocal space vector referred to one unit cell F*(h) the conjugate vector of F(h) F(h) modulus or amplitude of any vector F(h) f atomic scattering factor a, (3. Y angles between pairs of unit cell edges be, ac, and ab respectively V ABBREVIATIONS AMP adenosine 5'-monophosphate ATP adenosine 5'-triphosphate IMP inosine 5'-monophosphate GMP guanosine 5'-monophosphate GDP guanosine 5'-diphosphate GTP guanosine 5'-triphosphate HEPES N-2-hydroxyethylpiperazine-N'-2- ethanesulfonic acid MPD 2-methyl-2,4-pentanediol PEG 3350 polyethylene glycol, approximate molecular weight of 3350 pi isoelectric point 1 INTRODUCTION Adenylosuccinate synthetase catalyzes the first committed step toward the ^ novo biosynthesis of AMP (Figure 1). Adenylosuccinate is formed from the ligation of IMP and aspartate with the concomitant hydrolysis of GTP to GDP and P^. Adenylosuccinate lyase then cleaves adenylosuccinate releasing fumarate and AMP. Adenylate kinase transfers a high energy phosphate from ATP to AMP to produce two molecules of ADP. Finally, ATP is produced from ADP and P^ by oxidative phosphorylation in animals and by photophosphorylation in plants. OOCCHaÇHCOO- NH L-aspartate. GDP + PI Adenylosuccinate ribose-5'-P ribos6-5'-P synthetase Adenylosuccinate Inosine 5'-monophosphate Figure 1. The reaction catalyzed by adenylosuccinate synthetase Adenylosuccinate synthetase also participates in the purine nucleo­ tide cycle (1,2). This cycle (Figure 2) involves the interconversion of IMP, adenylosuccinate and AMP and is thought to play a number of regulatory roles including: 1) the liberation of ammonia from amino acids via aspartate, 2) regulation of adenine ribonucleotides, 3) regulation of phosphofructokinase activity and glycolysis via changes in AMP and ammonia levels, 4) regulation of phosphorylase b which is 2 activated by IMP, and 5) replenishment of citric acid cycle intermediates in tissues that do not have pyruvate carboxylase. "<X> ribose-5'-P IMP L-aspartate ^ / NH/ Adenylosuccinate synthetase f AMP deaminase / ^ GDPGDF + Pi OOCCHgCHCOO" NH NHg fumarate 6) adenylosuccinate "<ï> lyase ribose-5'-P ribose-S'-P AMP Adenylosuccinate Figure 2. The purine nucleotide cycle The enzyme has been isolated from plant (3), animal (4), and bacterial systems (4). The synthetase is believed to be enzymatically active as a dimer with typical dimeric molecular weights of 90,000 to 110,000 daltons (5-7). The enzyme from all sources requires a divalent cation for activity (7-11). Magnesium is the best activator, but Mn2+ 3 and Ca2+, and in some cases Co2+, Ba2+, and will substitute with decreased activity. Matsuda et al. (6) reported the isolation of two isozymic forms of adenylosuccinate synthetase. Later studies in chicken (12) and in healthy tissues of rat and rabbit also revealed only two distinct forms (8,13). They are distinguished by their isoelectric points. The acidic isozyme (pl=5.9) predominates in the brain, kidney, and spleen (6) and is believed to be involved primarily with ^ novo biosynthesis. The basic isozyme (pl=8.9) is found predominantly in skeletal and cardiac muscle and its primary role appears be the purine nucleotide cycle (1,2). The liver contains roughly equal amounts of both (6). This dissertation will concentrate on the structure of the synthetase isolated from Escherichia coli. A comprehensive review of the regulation, genetics and properties of the synthetase from various sources has been published (4). Properties of the Synthetase from Escherichia coli The synthetase from E. coli exists as a dimer with a molecular weight of 96,000 Da (14). The amino acid sequence has been determined (15). Post translational processing cleaves the N-terminal methionine leaving 431 amino acids in the polypeptide chain. Little homology exists between adenylosuccinate synthetase and other GTP binding proteins. This suggests the possibility of a unique nucleotide fold. The enzyme from ^ coli most closely resembles the acidic isozyme from mammalian tissues. 4 Mechanism COO- 0 fO_H- "OOCCHgCHCOOr B| 0 ^ 09 NH £-L-osp-GTP'IMP^= E-GOP-N'^N^ -L-asp^z^GDPPj-E- N^N. c R5P , R5P % //h 'OOCCHgÇHCOO" -OOCCHpCHCOO" -O^NH. r., -HO,P., I .ST.. St»-. ÂSP 5} ' R5P Figure 3. Three different reaction mechanisms proposed for adenylosuc­ cinate synthetase. This figure is taken from reference 4 Initial rate kinetic studies from a variety of sources (10,12,16-18)