Abstract：
:Two successive steps in de novo purine biosynthesis are catalyzed by the enzymes 5-aminoimidazole ribonucleotide (AIR) carboxylase and 4-[(N-succinylamino)carbonyl]-5-aminoimidazole ribonucleotide (SAICAR) synthetase. Amino acid sequence alignments of the proteins from various sources suggested that several unusual differences exist within the structure and function of these enzymes. In vertebrates, a bifunctional enzyme (PurCE) catalyzes successive carboxylation and aspartylation steps of AIR to form SAICAR. This is in contrast to the three proteins, PurK, PurE, and PurC, from Escherichia coli which have recently been shown to require 2 equiv of ATP for the AIR to SAICAR conversion in the presence of physiological HCO3- concentrations (Meyer et al., 1992). A comparative study of these proteins has been initiated using a high-production, heterologous expression system for the Gallus gallus AIR carboxylase-SAICAR synthetase and yields purified enzyme following a two-step procedure. Selective assays have been developed for all the enzymatic activities of the bifunctional protein. The G. gallus AIR carboxylase has no ATP dependence and displays a Km for HCO3- that is 10-fold lower than that for the related PurE protein from E. coli, supporting 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 of SAICAR synthetase with the affinity agent 5'-[4-(fluorosulfonyl)benzoyl]-adenosine (FSBA) and inhibition of AIR carboxylase with a tight-binding inhibitor 4-nitro-5-aminoimidazole ribonucleotide (NAIR). The native protein aggregates, and limited proteolysis indicates that the global structure of the protein involves two independent folding domains, each containing a different catalytic site.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Firestine SM,Davisson VJdoi
10.1021/bi00205a030subject
Has Abstractpub_date
1994-10-04 00:00:00pages
11917-26issue
39eissn
0006-2960issn
1520-4995journal_volume
33pub_type
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