PRYUVATE DEHYDROGENASE E1: STRUCTURE-FUNCTION STUDIES

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The overall objective of this research is to extend our knowledge of structure-function relationships in a large class of enzymes of fundamental importance to basic metablism; the thiamin diphosphate dependent alpha-keto acid decarboxylases. The project focuses on thiamin dependent enzymes that function within large multienzyme complexes, and their relationship to similar enzymes functioning in isolation. Despite their widespread importance in biochemistry and many years of study, only a single, recently obtained structural example exists for such a multienzyme complex components. Thus even the most basic structural information has just become available for any member of this family, and numerous questions remain regarding the catalytic, regulatory and assembly mechanisms. The goal is to provide the first high-resolution crystal structure analysis of a thiamin dependent E1 component from a pyruvate dehydrogenase multi-enzyme complex, PDHc E1 from E. coli, and compare it to other related enzymes. The structures it's to be compared with include that for pyruvate decarboxylase (PDC), which carries out essentially the same reaction on the same substrate but in isolation rather than in a complex, and that for the only other E1 structure for which data are available; the E1b component from a branched chain 2-oxoisovalerate dehydrogenase multienzyme complex. The latter (BDHcE1b) represents a different E1 class having no sequence homology, a different size and subunit composition, a different oligomeric state and operates on a different substrate than the E1 to be studied. X-ray diffraction methods also will be used to provide detailed information about structural changes resulting from interaction wth activators, substrates and inhibitors. The specific aims are: (1) to determine and analyze the structure of PDHc E1 to a resolution suitable for a complete chain trace; (2) To refine the complete structure to the highest resolution possible; (3) To compare th PDHc E1 structure with that for PDC; (4) To compare the PDHc E1 structure with that for BDHc E1b; (5) To identify amino acids in the PDHc E1 active site, assign functional roles to each and identify residues involved in lipoamide binding; (6) To identify binding sites and inhibition mechanisms for fluropyruvate, 2-oxo-3-butynoic acid, thiamin-2-thiothiazolone and monoclonal antibody 18A9; (7) To identify regulatory binding sites for GTP, Acetyl-CoA/CoA and probe pathways for information transfer to catalytic sites; (8) To analyze structures of complexes with covalently bound reaction intermediate analogs (phosphonate or phosphinate analogs of pyruvate).