title: Mechanism of action of Escherichia coli phosphoribosylaminoimidazolesuccinocarboxamide synthetase.
abstract: The conversion of ATP, L-aspartate, and 5-aminoimidazole-4-carboxyribonucleotide (CAIR) to 5-aminoimidazole-4-(N-succinylcarboxamide) ribonucleotide (SAICAR), ADP, and phosphate by phosphoribosylaminoimidazolesuccinocarboxamide synthetase (SAICAR synthetase) represents the eighth step of de novo purine nucleotide biosynthesis. SAICAR synthetase and other enzymes of purine biosynthesis are targets of natural products that impair cell growth. Prior to this study, no kinetic mechanism was known for any SAICAR synthetase. Here, a rapid equilibrium random ter-ter kinetic mechanism is established for the synthetase from Escherichia coli by initial velocity kinetics and patterns of linear inhibition by IMP, adenosine 5'-(beta,gamma-imido)triphosphate (AMP-PNP), and maleate. Substrates exhibit mutual binding antagonism, with the strongest antagonism between CAIR and either ATP or L-aspartate. CAIR binds to the free enzyme up to 200-fold more tightly than to the ternary enzyme-ATP-aspartate complex, but the latter complex may be the dominant form of SAICAR synthetase in vivo. IMP is a competitive inhibitor with respect to CAIR, suggesting the possibility of a hydrogen bond interaction between the 4-carboxyl and 5-amino groups of enzyme-bound CAIR. Of several aspartate analogues tested (hadacidin, l-malate, succinate, fumarate, and maleate), maleate was by far the best inhibitor, competitive with respect to L-aspartate. Inhibition by IMP and maleate is consistent with a chemical mechanism for SAICAR synthetase that parallels that of adenylosuccinate synthetase.
authors.affiliation: Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011, USA.
authors.forename: Scott W
authors.initials: SW
authors.lastname: Nelson
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Binkowski
authors.forename: Richard B
authors.initials: RB
authors.lastname: Honzatko
authors.forename: Herbert J
authors.initials: HJ
authors.lastname: Fromm
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000265:Adenylosuccinate Synthase / Q000737:chemistry / Q000378:metabolism; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D001224:Aspartic Acid / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D007291:Inosine Monophosphate / Q000737:chemistry; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008956:Models, Chemical; D010453:Peptide Synthases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012263:Ribonucleosides / Q000138:chemical synthesis; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D012263:Ribonucleosides; D007291:Inosine Monophosphate; D001224:Aspartic Acid; D000620:Aminoimidazole Carboxamide; D008345:Manganese; C011651:acadesine; D000255:Adenosine Triphosphate; D010453:Peptide Synthases; C083513:PurC protein, Bacteria; C022379:phosphoribosylaminoimidazole-succinocarboxamide synthetase; D000265:Adenylosuccinate Synthase; D008274:Magnesium