Carbamyl phosphate synthetase is an important housekeeping enzyme that provides carbamyl phosphate for arginine and pyrimidine biosynthesis. Our previous studies on the carbamyl phosphate synthetase genes of E. coli, yeast and rat indicated that despite differences in the substrate specificity, cofactor requirements, and subunit compositions of this phylogenetically diverse group of enzymes, they all have a common evolutionary origin. The present day arginine-specific synthetases are chimeric proteins composed of smaller modular units that endow the enzyme with a number of catalytic functions, including amide-nitrogen transfer and ATP dependent formation of C-N and C-O bonds. There are also compelling reasons to think that in some instances catalytic domains may have been modified and as a result acquired regulatory functions. In the present proposal, we intend to continue our studies of the arginine-specific carbamyl phosphate synthetases, the end objective being to describe the molecular mechanisms by which the catalytic and regulatory attributes of an evolutionarily and functionally related group of enzymes are modified and adapted in response to metabolic circumstances. Both genetic and recombinant DNA approaches will be used to achieve a dissection of the catalytic and regulatory sites of the enzyme. Most of these studies will employ mutants of E. coli impaired in carbamyl phosphate synthetase. A collection comprising 361 independent mutants has been isolated and is currently being analyzed. Carbamyl phosphate synthetases purified from strains expressing different growth requirements will be studied for their catalytic and regulatory properties. Mutations describing novel phenotypes will be mapped and sequenced to identify the catalytic and regulatory domains. Combined with in vitro mutagenesis of specific residues in the protein, this approach is anticipated to provide information about the regions that catalyze carbamate synthesis, convert carbamate to carbamyl phosphate, and bind the allosteric modifiers. A second set of studies will strive to identify the acetylglutamate regulatory domain in the rat enzyme. The coding sequence will De modified by deletions in the 5' coding region and expressed either in E. coli or in yeast. These experiments will test the hypothesis that a former glutaminase site has been converted to an acetylglutamate regulatory site.
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