The overall objective of this research is to very much enhance our understanding of structure-function relationships, including the mechanism and regulation of the yeast pyruvate decarboxylase. This enzyme requires thiamin diphosphate (the vitamin B1 coenzyme) and decarboxylates pyruvate in the penultimate step of alcohol fermentation. Funding is requested for the next period to continue research on this enzyme with the following major goals: (1) Further studies of the role of catalytic residues using a variety of spectroscopic methods. Determination of the rate-limiting steps in all active center variants, as well as wild-type enzyme, by direct assessment of the concentration of all key thiamin diphosphate-bound intermediates using intermediate partitioning and rapid-quench methods; (2) Experiments designed to identify the structural origins of and pathway for the 'alternating active sites in a functional dimer' mechanism proposed by the PI and coworkers for this enzyme, specifically by identifying the signal transduction pathway between catalytic centers. Experiments are designed to identify the structural origins of and pathway for dimer-dimer interactions leading to the two different conformations observed by the X-ray studies. In particular, the Pl's group has recently reported kinetics studies indicating that there are two active conformations of the enzyme, one regulated by substrate, the other is not. It is important then to further delineate the structural basis for these two types of conformations and those constructs that can be crystallized will be subjected to high-resolution X-ray methods in an ongoing collaboration with William Furey, Univ. of Pittsburgh, School of Medicine. Goals 1 and 2 will be heavily dependent on the Pl's ability to not only introduce any desired substitution, but also to create tetramers with any desired composition of modified subunits. This enzyme continues to provide an outstanding paradigm not only for thiamin-dependent enzymes, but also for interaction of active centers in homo-tetrameric enzymes. ? ?
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