CARBOXYLASES IN DE-NOVO PURINE BIOSYNTHESIS - CHARACTERIZATION OF THEGALLUS-GALLUS BIFUNCTIONAL ENZYME

Two successive steps in de novo purine biosynthesis are catalyzed by t he enzymes 5-aminoimidazole ribonucleotide (AIR) carboxylase and 4-[(N -succinylamino)carbonyl]-5-aminoimidazole ribonucleotide (SAICAR) synt hetase. Amino acid sequence alignments of the proteins from various so urces suggested that several unusual differences exist within the stru cture and function of these enzymes. In vertebrates, a bifunctional en zyme (PurCE) catalyzes successive carboxylation and aspartylation step s of AIR to form SAICAR. This is in contrast to the three proteins, Pu rK, PurE, and PurC, from Escherichia coli which have recently been sho wn to require 2 equiv of ATP for the AIR to SAICAR conversion in the p resence of physiological HCO3- concentrations (Meyer et al., 1992). A comparative study of these proteins has been initiated using a high-pr oduction, heterologous expression system for the Gallus gallus AIR car boxylase-SAICAR synthetase and yields purified enzyme following a two- step procedure. Selective assays have been developed for all the enzym atic activities of the bifunctional protein. The G. gallus AIR carboxy lase has no ATP dependence and displays a K-m for HCO3- that is 10-fol d lower than that for the related PurE protein from E. coli, supportin g the hypothesis that the two enzymes require different substrates. No common cofactors or metals are required for catalysis. Each catalytic activity has been shown to be independent by selective inactivation o f SAICAR synthetase with the affinity agent 5'-[4-(fluorosulfonyl)benz oyl]-adenosine (FSBA) and inhibition of AIR carboxylase with a tight-b inding inhibitor 4-nitro-5-aminoimidazole ribonucleotide (NAIR). The n ative protein aggregrates, and limited proteolysis indicates that the global structure of the protein involves two independent folding domai ns, each containing a different catalytic site.