PMID:15641804

Citation	Nelson, SW, Binkowski, DJ, Honzatko, RB and Fromm, HJ (2005) Mechanism of action of Escherichia coli phosphoribosylaminoimidazolesuccinocarboxamide synthetase. Biochemistry 44:766-74
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.
Links	PubMed Online version:10.1021/bi048191w
Keywords	Adenosine Triphosphate/metabolism; Adenylosuccinate Synthase/chemistry; Adenylosuccinate Synthase/metabolism; Aminoimidazole Carboxamide/analogs & derivatives; Aminoimidazole Carboxamide/chemical synthesis; Aspartic Acid/metabolism; Bacterial Proteins/chemistry; Bacterial Proteins/genetics; Chromatography, High Pressure Liquid; Cloning, Molecular; Enzyme Inhibitors/chemistry; Escherichia coli Proteins/antagonists & inhibitors; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/genetics; Escherichia coli Proteins/isolation & purification; Hydrogen-Ion Concentration; Inosine Monophosphate/chemistry; Kinetics; Magnesium/metabolism; Manganese/metabolism; Models, Chemical; Peptide Synthases/antagonists & inhibitors; Peptide Synthases/chemistry; Peptide Synthases/genetics; Peptide Synthases/isolation & purification; Recombinant Proteins/antagonists & inhibitors; Recombinant Proteins/chemistry; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism; Ribonucleosides/chemical synthesis; Substrate Specificity
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