Pyruvate dehydrogenase complex provides an important metabolic link between glycolysis nd the complete oxidation of glucose. zit commits pyruvate to oxidation via acetyl-CoA and Krebs Cycle since there is no biochemical means by which acetyl-CoA can be converted back to pyruvate. The activity of pyruvate dehydrogenase complex is controlled by reversible phosphorylation. Evidence obtained just recently indicates that the system responsible for covalent modification of pyruvate dehydrogenase is organized in a much more complex way than was believed before. It appears that mammalian pyruvate dehydrogenase kinase responsible for phosphorylation and inactivation of pyruvate dehydrogenase i) belongs to a new family of eukaryotic serine-specific protein kinases, ii) exists as two isoenzymic forms different in terms of primary structure and tissue distribution, and iii) may be a subject of regulation by phosphorylation- dephosphorylation itself. The current proposal is designed to continue the studies on the regulation of pyruvate dehydrogenase in directions that follow form these new data. It targets characterization of the catalytic mechanism of pyruvate dehydrogenase kinase, the determination of the regulatory properties of the two isoforms of the kinase, and the elucidation of the role of autophosphorylation of the first isoform of pyruvate dehydrogenase kinase in regulation of its activity. The detailed specific aims of this proposal are; 1) to identify the amino acid residues within the catalytic domain of pyruvate dehydrogenase kinase important for kinase function: 2) to characterize the regulatory properties of the two isoforms of pyruvate dehydrogenase kinase; and 3) to characterize the role of autophosphorylation of serine residues in the regulation of pyruvate dehydrogenase kinase activity. The accomplishment of these goals will not only bring nre insight into our understanding of the structure and function of pyruvate dehydrogenase complex, but will also allow investigators to move further toward characterization of the molecular mechanisms responsible for i) selection respiratory fuels in mammals during starvation, ii) tissue specific differences in regulation of aerobic oxidation of glucose, and iii) abnormal regulation of pyruvate dehydrogenase complex in pathogenesis of diabetes, cancer, sepsis, cardiomyopathy and obesity.
