PYRUVATE DECARBOXYLASE STRUCTURE/FUNCTION RELATIONSHIPS

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The overall objective of this research is to substantially extend our knowledge of structure-function relationships in a class of enzymes of fundamental importance in metabolism; the alpha-keto acid decarboxylases. Despite years of study and their widespread importance in basic biochemistry, at present there isn't a single structural example available for thiamin diphosphate (TDP) dependent enzymes of any sort, and numerous structure related questions remain regarding the catalytic mechanism and regulation. Our goal is to provide detailed information about the basic subunit (monomer) and holoenzyme (tetramer, including TDP and Mg+2 cofactors) structures for the most widely studied TDP dependent enzyme (pyruvate decarboxylase, PDC). This will be achieved by determining the 3D structure of the holoenzyme complex via single crystal x-ray diffraction methods. Information regarding the catalytic mechanism and regulation will then be obtained by comparing/contrasting the native structure with the structure in the presence of several mechanism based inhibitors and/or regulating compounds. The specific aims are: (1) to determine the structure of the holoenzyme PDC such that the complete fold (chain trace) can be obtained; (2) to complete and refine the structure to 2.4alpha resolution; (3) to probe the relative roles of the enzyme and cofactors in catalysis; (4) to determine the number of, and locations for all cofactors, and to support/disprove a recent hypothesis regarding a common TDP binding site in all TDP dependent enzymes; (5) to identify the conformation of TDP in the presence and absence of substrate; (6) to identify and explain the roles of specific amino acids affecting cofactor binding and activity, but not involved in catalysis; (7) to identify the residues involved in substrate binding, and binding at the regulatory site; (8) to examine the structure when "frozen" at various stages in the reaction pathway; (9) to compare and contrast the results with a related, but functionally distinct enzyme (transketolase) once both coordinate sets become available.