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Kinetic Studies of Escherichia Coli and Human SAICAR Synthetase Daniel Joseph Binkowski Iowa State University

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Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 2007 Kinetic studies of Escherichia coli and human SAICAR synthetase Daniel Joseph Binkowski Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Biochemistry Commons Recommended Citation Binkowski, Daniel Joseph, "Kinetic studies of Escherichia coli and human SAICAR synthetase" (2007). Retrospective Theses and Dissertations. 15779. https://lib.dr.iastate.edu/rtd/15779 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]. Kinetic studies of Escherichia coli and human SAICAR synthetase by Daniel Jospeh Binkowski A dissertation submitted to the graduate faculty in partial fulfillment of the requirement for the degree of DOCTOR OF PHILOSOPHY Major: Biochemistry Program of Study Committee Richard B. Honzatko, Co-major Professor Herbert J. Fromm, Co-major Professor Reuben J. Peters Alan A. Dispirito Drena L. Dobbs Iowa State University Ames, Iowa 2007 Copyright © Daniel Joseph Binkowski, 2007. All rights reserved. UMI Number: 3383365 INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. ______________________________________________________________ UMI Microform 3383365 Copyright 2009 by ProQuest LLC All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. _______________________________________________________________ ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, MI 48106-1346 ii TABLE OF CONTENTS Abbreviations iv Abstract vi Chapter I. General Introduction 1 Introduction 1 Thesis Organization 5 References 6 Figures and Tables 9 Chapter II. Mechanism of action of Escherichia coli Phosphoribosylaminoimidazolesuccinocarboxamide synthetase 17 Abstract 17 Introduction 18 Experimental Procedures 20 Results 23 Discussion 29 References 32 Figures and Tables 36 Chapter III. Nucleotide complexes of Escherichia coli Phosphoribosylaminoimidazolesuccinocarboxamide synthetase 42 Abstract 42 Introduction 43 Experimental Procedures 44 Results 48 Discussion 52 References 55 Figures and Tables 60 Chapter IV. Recognition of L-Aspartate by Phosphoribosylaminoimidazolesuccinocarboxamide Synthetase Involves 5-Amino-4-carboxyimidazole Ribonucleotide 70 Abstract 70 Introduction 71 Experimental Procedures 73 Results 77 Discussion 80 References 84 Figures and Tables 88 Chapter V. Linkage of Function in Human AIR Carboxylase/SAICAR Synthetase 96 iii Abstract 96 Introduction 97 Experimental Procedures 100 Results 104 Discussion 110 References 114 Figures and Tables 117 Chapter VI. General Conclusions and Future Work 124 iv Abbreviations AIR 5-amino-imidazole- ribonucleotide AMP, ADP, ATP adenosine mono-, di-, triphosphate CAIR 5-aminoimidazole-4-carboxy ribonucleotide DNPS de novo Purine Biosynthesis E. Coli Escherichia Coli eSS E. coli SAICAR synthetase GMP, GDP, GTP Guanosine mono-, di-, triphosphate hSS Human SAICAR synthetase/AIR Carboxylase IMP Inosine monophosphate Kcat Catalytic constant KI Inhibition dissociation constant Km Michaelis constant NADH Nicatinamide adenine dinucleotide (reduced) 1-PIMP 1- phosphoryl-IMP N5-CAIR N5-Carboxy-5-aminoimidazole ribonucleotide 6-PIMP 6-phosphoryl-IMP PIX Positional Isotope Exchange PK/LDH Pyruvate Kinate, Lactate dehydrogenase PRPP 5-phospho-α-D-ribosyl-1-pyrophosphate SAICAR 5-aminoimidazole-4(N-succinylcarboxamide) ribonucleotide SAMP Succinyl-AMP tSS Thermatoga Maritima SAICAR Synthetase v ySS Yeast SAICAR synthetase vi Abstract Phosphoribosyl-aminoimidazole-succinocarboxamide synthetase (SAICAR synthetase) catalyzes the eighth step of de novo IMP biosynthesis in bacteria, and the seventh step in humans. The SAICAR synthetase reaction is analogous to that of adenylosuccinate synthetase (first committed step of AMP biosynthesis), using ATP to ligate 5-amino-imidazole-4-carboxy ribonucleotide and L- aspartate to produce 5-aminoimidazole-4(N-succinylcarboxamide) ribonucleotide (SAICAR). SAICAR synthetase and other enzymes of purine nucleotide biosynthesis are targets of natural products that impair cell growth. Prior to these studies, the kinetic mechanism of any SAICAR synthetase was unknown. Herein are reported the kinetic mechanisms of bacterial and human SAICAR synthetase activities, the structure of the bacterial enzyme from Escherichia coli, and substrate recognition properties of both the human and bacterial forms of the enzyme. The human enzyme is bifunctional, combining 5-aminoimidazole ribonucleotide (AIR) carboxylase activity with SAICAR synthetase activity. A determination of the kinetic mechanism of SAICAR synthetase requires the absence of AIR carboxylase activity. A slow, tight-binding inhibitor (4-Nitro-5- aminoimidazole ribonucleotide) and the mutation of a residue critical to catalysis independently eliminated interfering AIR carboxylase activity. The kinetic mechanism of the SAICAR synthesis is the same for the two forms of AIR carboxylase-impaired enzyme, but differences in kinetic parameters demonstrate a linkage mechanism between the two types of active site in the human bifunctional enzyme. Human SAICAR synthetase may impose a metering function that insures constant output of SAICAR over a ten-fold variation in the concentration of CAIR. 1 Chapter I. Introduction Phosphoribosyl-aminoimidazole-succinocarboxamide synthetase [EC 6.3.2.6, 5’- phosphoribosyl-4carboxy-5-aminoimidazole: L-aspartate ligase (ADP)] (SAICAR synthetase), an essential enzyme in de novo purine biosynthesis, catalyzes the conversion of ATP, 5-aminoimidazole- 4-carboxy ribonucleotide (CAIR) and L-aspartate to 5-aminoimidazole-4(N-succinylcarboxamide) ribonucleotide (SAICAR), ADP and Pi. In bacteria such as Escherichia coli, the conversion of 5- aminoimidazole ribonucleotide (AIR) into SAICAR employs three single-function enzymes, PurK (AIR to N5-CAIR), PurE (N5-CAIR to CAIR), and PurC (CAIR to SAICAR)(1), whereas in vertebrates, a single enzyme catalyzes the transformation of AIR to SAICAR (2, 3). The chemistry of the AIR to SAICAR transformation differs for vertebrate and bacterial systems, the latter using ATP and bicarbonate in the generation of N5-CAIR, whereas the vertebrate enzyme sequesters and uses carbon dioxide and AIR directly to make CAIR (4-9). Fungi such as Candida albicans and Cryptococcus neoformans combine the PurK and PurE functions into a single protein, but retain a single-function enzyme, similar to that of E. coli enzyme, for the synthesis of SAICAR from CAIR (Figure 1). This enzyme is the eighth step of DNPS in E. coli and the seventh in humans, and is also the last step in IMP biosynthesis that requires ATP. Enzymes of de novo purine nucleotide biosynthesis are targets in the treatment of cancer and infectious disease. Many cancers have damaged salvage pathways for purine nucleotides because of methylthioadenosine phosphorylase (MTAP) deficiencies, and hence are more sensitive to the inhibition of de novo purine nucleotide biosynthesis than non-cancerous cells (roughly 30% of all T- cell acute Lymphocytic Leukemia cases, for example) (12) (Figure 2). More recently, blockage of purine metabolism in Candida albicans and Crypotcoccus neoformans attenuates virulence in invasive infections and meningoenciphilitis infections, respectively, of immune compromised rodent models, even though these organisms are capable of salvaging purines from the host (11, 13). Hence, the study of SAICAR synthetase from E. coli and mammals pertains directly to development of drugs 2 and strategies in the treatment of cancers containing MTAP deficiencies, as well as treatments of immune compromised individuals dealing with opportunistic bacterial infections. Efforts to discover inhibitors or anti-metabolites of different steps of purine nucleotide biosynthesis have brought forth no inhibitors of SAICAR synthetase, but the synthetase does transform at least one substrate analogs into a potent inhibitor. L-Alanosine, shows promise for treatment of certain types of leukemia and gliomas (12). L-Alanosine and L-aspartate have indistinguishable kinetic parameters in reactions catalyzed by the E. coli synthetase; a property not observed for other systems (aspartate carbamoyltransferase or adenylosuccinate synthetase) that use L-aspartate as a substrate (14-16). SAICAR synthetase in humans uses L-alanosine in place of L- aspartate (albeit inefficiently), forming the SAICAR analog, 5-aminoimidazole-4(N- alanosylcarboxamide) ribonucleotide, a potent inhibitor of adenylosuccinate synthetase and adenylosuccinate lyase (Figure 3). Treatment with L-alanosine when supplemented with deoxyadenosine is toxic to only cancerous cells, thus allowing for the selective inhibition of cancer cell lines containing homozygous methylthioadenosine
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  • Pairs of Dipeptides Synergistically Activate the Binding of Substrate by Ubiquitin Ligase Through Dissociation of Its Autoinhibitory Domain

    Pairs of Dipeptides Synergistically Activate the Binding of Substrate by Ubiquitin Ligase Through Dissociation of Its Autoinhibitory Domain

    Pairs of dipeptides synergistically activate the binding of substrate by ubiquitin ligase through dissociation of its autoinhibitory domain Fangyong Du*, Federico Navarro-Garcia†, Zanxian Xia, Takafumi Tasaki, and Alexander Varshavsky‡ Division of Biology, California Institute of Technology, Pasadena, CA 91125 Contributed by Alexander Varshavsky, August 29, 2002 Protein degradation by the ubiquitin (Ub) system controls the con- S. cerevisiae N-end rule pathway is the control of peptide import centrations of many regulatory proteins. The degradation signals through regulated degradation of the 35-kDa homeodomain pro- (degrons) of these proteins are recognized by the system’s Ub ligases tein CUP9, a transcriptional repressor of the di- and tripeptide (complexes of E2 and E3 enzymes). Two substrate-binding sites of transporter PTR2 (13, 24, 25). CUP9 contains an internal (C UBR1, the E3 of the N-end rule pathway in the yeast Saccharomyces terminus-proximal) degron which is recognized by a third substrate- cerevisiae, recognize basic (type 1) and bulky hydrophobic (type 2) binding site of UBR1. Previous work (13) has shown that the N-terminal residues of proteins or short peptides. A third substrate- UBR1-dependent degradation of CUP9 is allosterically activated by binding site of UBR1 targets CUP9, a transcriptional repressor of the dipeptides with destabilizing N-terminal residues. In the resulting peptide transporter PTR2, through an internal (non-N-terminal) de- positive feedback, imported dipeptides bind to UBR1 and accel- gron of CUP9. Previous work demonstrated that dipeptides with erate the UBR1-dependent degradation of CUP9, thereby dere- destabilizing N-terminal residues allosterically activate UBR1, leading pressing the expression of PTR2 and increasing the cell’s capacity to accelerated in vivo degradation of CUP9 and the induction of PTR2 to import peptides (13).
  • The Structure of Carbamoyl Phosphate Synthetase Determined to 2.1 A

    The Structure of Carbamoyl Phosphate Synthetase Determined to 2.1 A

    research papers Acta Crystallographica Section D Biological The structure of carbamoyl phosphate synthetase Crystallography determined to 2.1 AÊ resolution ISSN 0907-4449 James B. Thoden,a Frank M. Carbamoyl phosphate synthetase catalyzes the formation of Received 10 March 1998 Raushel,b Matthew M. Benning,a carbamoyl phosphate from one molecule of bicarbonate, two Accepted 29 April 1998 2+ Ivan Raymenta and Hazel M. molecules of Mg ATP and one molecule of glutamine or a ammonia depending upon the particular form of the enzyme PDB Reference: carbamoyl Holden * phosphate synthetase, 1jdb. under investigation. As isolated from Escherichia coli, the enzyme is an , -heterodimer consisting of a small subunit aInstitute for Enzyme Research, The Graduate that hydrolyzes glutamine and a large subunit that catalyzes School, and Department of Biochemistry, the two required phosphorylation events. Here the three- College of Agricultural and Life Sciences, University of Wisconsin±Madison, 1710 dimensional structure of carbamoyl phosphate synthetase University Avenue, Madison, Wisconsin 53705, from E. coli re®ned to 2.1 AÊ resolution with an R factor of USA, and bDepartment of Chemistry, Texas 17.9% is described. The small subunit is distinctly bilobal with A&M University, College Station, Texas 77843, a catalytic triad (Cys269, His353 and Glu355) situated USA between the two structural domains. As observed in those enzymes belonging to the = -hydrolase family, the active-site Correspondence e-mail: nucleophile, Cys269, is perched at the top of a tight turn. The [email protected] large subunit consists of four structural units: the carboxypho- sphate synthetic component, the oligomerization domain, the carbamoyl phosphate synthetic component and the allosteric domain.