The conversion of aminoimidazole ribonucleotide (AIR) to carboxyaminoimidazole ribonucleotide (CAIR) by phosphoribosylaminoimidazole carboxylase (AIR carboxylase) and the conversion of conversion of ATP,

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-aspartate and CAIR to 5-aminoimidazole-4-(N-succinylcarboxamide) ribonucleotide (SAICAR), ADP and phosphate by phosphoribosylaminoimidazolesuccinocarboxamide synthetase (SAICAR synthetase) represent the 7^th and 8^th steps respectively of de novo purine nucleotide biosynthesis. For the vertebrate enzyme system, AIR carboxylase and SAICAR synthetase are combined in a bifunctional protein encoded by a single gene. Here, a porcine gene encoding AIR carboxylase/SAICAR synthetase was cloned and expressed in Escherichia coli. The native molecular weight of the purified recombinant protein infers an octameric assembly of subunits. Site-directed mutagenesis has identified residues (Lys19, Asp277, and Lys304) critical to AIR carboxylase or SAICAR synthetase activities, indicating a protein defined by separate domains for each catalytic function. The decline in the K _m for CAIR in the SAICAR synthetase reaction due to mutations in the AIR carboxylase active site infers some level of active-site coupling in the oligomeric vertebrate enzyme. A double mutant Lys³⁰⁴→Ala/Val ²⁰⁸→Glu exhibits CAIR-dependent ATPase activity in the absence of L-aspartate, suggesting that CAIR binding induces a conformational change in the ATP binding pocket and causes the hydrolysis of ATP or the hydrolysis of the presumed carbonyl phosphate intermediate of CAIR.