REACTIONS CATALYZED BY 5-AMINOIMIDAZOLE RIBONUCLEOTIDE CARBOXYLASES FROM ESCHERICHIA-COLI AND GALLUS-GALLUS - A CASE FOR DIVERGENT CATALYTIC MECHANISMS

A comparative investigation of the substrate requirements for the enzyme 5-aminoimidazole ribonucleotide (AIR) carboxylase from E. coli and G. gallus has been conducted using in vivo and in vitro studies. In Escherichia coli, two enzymes PurK and PurE are required for the transformation of AIR to 4-carboxy-5-aminoimidazole ribonucleotide (CAIR). The Gallus gallus PurCE is a bifunctional enzyme containing AIR carboxylase and 4-[(N-succinylamino)carbonyl]-5-aminoimidazole ribonucleotide (SAICAR) synthetase. The E. coli PurE and the C-terminal domain of the G. gallus PurCE protein maintain a significant degree of amino acid sequence identity and also share CAIR as a product of their enzymatic activities. The substrate requirements of AIR carboxylases from E. coli and G. gallus have been compared by a series of in vitro experiments. The carbamic acid, N-5-carboxyaminoimidazole ribonucleotide (N-5-CAIR) is a substrate for the E. coli PurE (Mueller et al., 1994) but not for the G. gallus AIR carboxylase. In contrast, AIR and CO2 are substrates for the G. gallus AIR carboxylase. The recognition properties of the two proteins were also compared using inhibition studies with 4-nitro-5-aminoimidazole ribonucleotide (NAIR). NAIR is a tight-binding inhibitor of the G. gallus AIR carboxylase (K-i = 0.34 nM) but only a steady-state inhibitor (K-i = 0.5 mu M) of the E. coli PurE. These data suggest significant differences in the transition states for the reactions catalyzed by these two evolutionarily related enzymes. Using separate, constitutive overexpression systems for E. coli purK or purE, or G. gallus purCE, the impact of each of these enzymes upon the growth rates of a PurK-deficient strain of E. coli was evaluated. The results suggest that a PurK deficiency effects purine metabolism by creating a rate-limiting chemical carboxylation of AIR. Heterologous expression of the G. gallus PurCE overcomes this limitation by providing a different pathway for conversion of AIR to CAIR.