Acetyl-CoA carboxylase catalyzes a rate limiting and commited reaction in the de novo synthesis of palmitate from acetyl-CoA. This enzyme is subject to regulation by allosteric modification, covalent phosphorylation and dephosphorylation, and transcriptional control at the gene level. Diet and hormones such as insulin, glucagon and epinephrine play a key role in the regulation of this enzyme. Native carboxylase consists of two multifunctional subunits, each containing domains for catalytic and regulatory activities, as well as a biotin binding site. The organization of these domains on the subunit polypeptide will be determined and related to the amino and carboxyl ends of the polypeptide. Selected proteases will be employed in these studies and a linear proteolytic map of the carboxylase subunit will be contructed. This map will be used as a reference point for the localization of active centers within the polypeptide. In these studies a combination of active site reagents, immunological probes and direct isolation of enzymatically active fragments will be employed. The citrate activation and palmitoyl-CoA inhibition sites and the phosphorylation sites are among those that will be assigned on the activity map. These sites will be identified in part by using photolabile derivatives, radiolabeled substrates or a combination thereof. The orientation of the subunits in the dimer form will be determined by electron microscopy of negatively stained avidin-carboxylase complexes. The orientation of the biotinyl group will be established and the question of half- vs full-site activity of the dimer will be addressed. The regulation of the carboxylase by phosphorylation and dephosphorylation will be studied in detail and the kinases and phosphatases involved will be isolated and characterized. The protein substrate specificity of the kinases and phosphatases will be determined and compared with known kinases and phosphatases. The regulation of the kinases and phosphatases will be investigated and the effect of hormones (insulin, glucagon, epinephrine), cyclic nucleotides and diet on these regulatory enzymes will be studied. Clones containing DNA complementary to carboxylase mRNA will be selected from our cDNA banks and utilized in studies of regulation of carboxylase mRNA. The cDNA will be used in determining the amino acid sequence of the carboxylase subunit and in the isolation and characterization of the carboxylase gene.
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