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 enzy me 5-aminoimidazole ribonucleotide (AIR) carboxylase from E. coli and G. gallus has been conducted using in vivo and in vitro studies. In Es cherichia coil, two enzymes PurK and PurE are required for the transfo rmation of AIR to 4-carboxy-5-aminoimidazole ribonucleotide (CAIR). Th e Gallus gallus PurCE is a bifunctional enzyme containing AIR carboxyl ase 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 s equence identity and also share CAIR as a product of their enzymatic a ctivities. 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 f or the G. gallus AIR carboxylase. In contrast, AIR and CO2 are substra tes for the G. gallus AIR carboxylase. The recognition properties of t he two proteins were also compared using inhibition studies with 4-nit ro-5-aminoimidazole ribonucleotide (NAIR). NAIR is a tight-binding inh ibitor of the G. gallus AIR carboxylase (K-i = 0.34 nM) but only a ste ady-state inhibitor (K-i = 0.5 mu M) of the E. coli PurE. These data s uggest significant differences in the transition states for the reacti ons catalyzed by these two evolutionarily related enzymes. Using separ ate, constitutive overexpression systems for E. coli purK or purE, or G. gallus purCE, the impact of each of these enzymes upon the growth r ates 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 differ ent pathway for conversion of AIR to CAIR.