The overall objective of this research is to enhance our understanding of structure-function relationships, including the mechanism and regulation of the El component of the pyruvate dehydrogenase multienzyme complex isolated from E. coli. This enzyme requires thiamin diphosphate (the vitamin B coenzyme) and decarboxylates pyruvate, the product of glycolysis, to initiate a series of reactions, a major product being acetyl-Coenzyme A, the starting material for the citric acid cycle. Among the specific goals for the requested period are: (1) Production of wild-type and variant El enzymes (the variants being made by site directed mutagenesis of the plasmid specifically bearing only the gene coding for El) for both mechanistic and structural studies, including, determination of the X-ray structure in a collaboration wit William Furey, University of Pittsburgh (with whom the principal investigator has collaborated in the solution of the simpler yeast pyruvate decarboxylase structure); (2) Experiments designed to a. identify the amino acids responsible for catalysis, and assign the function in catalysis; b. study the fate of key thiamin-bound intermediates on the enzyme; c. study the rate-limiting steps in catalysis and the structure of transition states in wild-type and active center variant enzymes; d. identify the site of, and solve the mechanism of inactivation of El by substrate fluoropyruvate, 2-oxo-3-butynoic acid (a potent substrate analog inhibitor developed in the principal investigator's laboratory), a putative transition-state analog, and several highly inhibitory monoclonal antibodies (also developed in the principal investigator's laboratory); (3) Experiments to identify the site(s) at which a variety of metabolic regulators interact with the El, including the cofactors and the products of the reaction, as well as GTP- and the pathway by which the information i transmitted to the active center; and (4) Explore the principal investigator's hypothesis concerning the key reductive acetyl transfer between the El and E2 enzymes. Since this enzyme is at a key metabolic junction, the principal investigator believes that in the coming years he and his collaborators have an opportunity to contribute to a better molecular-level understanding of this member of a very large class of related multienzyme complexes.
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