This proposal is concerned with delineation of remaining structural and mechanistic questions on pyruvate decarboxylase (PDC, E.C. 4. 1. 1. 1), perhaps the simplest nonoxidative decarboxylase requiring thiamin diphosphate (ThDP, the vitamin B1 coenzyme, of fundamental significance in human metabolism). The brewer' yeast enzyme was recently purified as fully active alpha4 and beta4 homotetramers and the alpha4 structure was crystallized by the PI and collaborators at the VA Hospital in Pittsburgh pointing to the likely success of resolution to at least 2.4 angstroms. With the evidence already accumulated from the 3 angstroms map of the enzyme, for the first time on this representative of a large group of alpha-keto acid decarboxylases, rational experiments can be designed to probe: a. the conformation of the enzyme-bound coenzyme in the absence of substrate, as well as in the three covalent substrate-coenzyme complexes invoked, based on chemical analogy; b. the chemical behavior in the absence and presence of substrate of the two aromatic rings (thiazolium and 4-aminopyrimidine), and test of the hypothesis that both rings ( not only the thiazolium) are participants in catalysis; c. the environment of the coenzyme and the function of amino acids surrounding it in catalysis; and d. the effect of pyruvamide, a nondecarboxylatable substrate surrogate that is capable of shifting the enzyme from a low to a high activity form, on structure and mechanism. Most prominent among the tools to address these questions will be : a. x-ray crystallographic methods (this part of the research is a collaborative effort with Furey, Sax and coworkers at the VA Hospital Biocrystallography Lab/ Univ. of Pittsburgh) ; b. site-directed mutagenesis of amino acids found near the catalytic and regulatory sites based on the x-ray crystallographic and mechanistic information; c. further elucidation of the chemistry and enzyme-bound environment of the enamine, one of the three ThDP-substrate covalent complexes on PDC; d. multinuclear magnetic resonance on ThDP bound to PDC, with ThDP labelled at the C2, Nl' and N4' atoms to provide information concerning the state of ionization, and the charge densities of the coenzyme during the catalytic sequence in the absence and in the presence of regulators;. and 5. modeling based on the x-ray coordinates to help refine mechanistic models, to help design even more insightful experiments, and to help construct three dimensional models of two enzymes with very high sequence homology to PDC's (pyruvate oxidase and acetolactate synthetase) that have not been crystallized yet, and that have identical mechanisms through pyruvate decarboxylation, but diverge significantly thereafter. Undoubtedly, the fundamental questions to be resolved by the proposed research, will be of profound interest and significance to many other research groups working on thiamin around the globe.
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