(From the application abstract:) The mitochondrial pyruvate dehydrogenase complex (PDC) catalyzes the oxidative decarboxylation of pyruvate, the reaction that determines the metabolic fate of carbohydrates. The enzymatic activity of the mammalian PDC is regulated by reversible phosphorylation. The specific kinase (pyruvate dehydrogenase kinase or PDK) converts it to an inactive form that can be reactivated only by a specific phosphatase. The hyperphosphorylation of PDC observed in diabetes, ischemia, and metabolic acidosis directly contributes to the morbidity and mortality associated with these conditions. It is generally believed that the hyperphosphorylation is due, in part, to enhanced kinase activity. Recently this laboratory provided the first data indicating that, in humans and other mammals there are multiple isoenzymes of PDK. The physiological significance of multiple isoenzymes is currently unknown. The results available thus far strongly suggest that the isoenzymes are functionally different. The isoenzyme PDK2 is likely to be responsible for the * short-term regulation of PDC activity. The inducible isoenzyme PDK4, in contrast, may be mainly responsible for long-term control. Its over-expression in diabetes is likely a leading cause of the hyperphosphorylation of PDC that, in turn, contributes to hyperglycemia. This proposal is aimed to further elucidate the structure, function, regulation and physiological significance of the multiple isoenzymes of PDK. Its major goals are: 1) to determine the three dimensional structure of pyruvate dehydrogenase kinase; 2) to elucidate the molecular basis for catalysis and substrate recognition by pyruvate dehydrogenase kinase; 3) to further define the molecular mechanisms responsible for regulation of pyruvate dehydrogenase kinase activity; 4) to characterize the molecular interactions between isozymes, as well as between isozymes and pyruvate dehydrogenase complex under normal conditions, as well as under starvation and diabetes. These goals will be achieved though a combination of structure/functional analysis, biochemical characterization, as well as more physiologically oriented studies of isozymes under conditions such as starvation and diabetes. This will allow us to understand how this structurally unique protein kinase functions. It will also allow us to take the first step towards the design of isoenzyme- specific drugs that may alleviate some of the symptoms and prevent complications associated with diabetes, ischemia and acidosis.
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