This research project is directed toward elucidating the structure, function and regulation of the alpha-keto acid dehydrogenase multienzyme complexes from eukaryotic,and prokaryotic cells. During the next five-year period emphasis will be placed on structure-function studies of eukaryotic pyruvate dehydrogenase (PDH) complexes, particularly the PDH complex from Saccharomyces cerevisiae, using biochemical, molecular genetic, and X-ray crystallographic approaches.
The specific aims are (1) to conduct structure-function Analyses, including collaborative X-ray crystallography, of S. cerevisiae dihydrolipoamide acetyltransferase (E 2p) and its structural domains using recombinant proteins; (2) to continue to elucidate the nature and function of the protein X component of the yeast PDH complex, using biochemical and molecular genetic approaches; (3) to conduct collaborative X-ray crystallographic analysis and site-directed mutagenesis of dihydrolipoamide dehydrogenase (E3); (4) to clone the yeast gene encoding the beta subunit of pyruvate dehydrogenase (El) and overexpress E1alpha and E1beta for functional studies; (5) to purify to homogeneity and further characterize the protein kinase which modulates the activity of the bovine branched-chain alpha-keto acid dehydrogenase complex; (6) to clone and sequence cDNAs encoding the two subunits of the regulatory enzyme PDH phosphatase. The alpha-keto acid dehydrogenase multienzyme complexes play central roles in cellular metabolism, are major sites of regulation, and are clinically important. In !mammalian cells, these multienzyme complexes are located within mitochondria, an organelle that is the major source of energy for cellular functions. Regulation of the flux of carbon through the PDH complex plays an essential role in the control of energy metabolism. PDH phosphatase is one of the target enzymes of insulin action. The branched-chain alpha-keto acids represent an important source of carbon for cellular energy generation, carbohydrate biosynthesis, and ketone body production in various tissues. Elucidation of the structure and catalytic and regulatory properties of the alpha-keto acid dehydrogenase complexes and their component enzymes is a necessary prerequisite to understanding the in vivo regulation of their activities. Inherited and acquired disorders of pyruvate and branched-chain amino acid metabolism have been reported. Analysis of the etiology of these disorders rests on knowledge of normal metabolism of 'the alpha-keto acids.
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