The overall objective of this research is to very much enhance our knowledge of the structure-function relationships and mechanism of a class of enzymes of fundamental importance to basic metabolism, the thiamin diphosphate-dependent 2-oxoacid decarboxylases. With the X-ray structure of a member of this class pyruvate decarboxylase (PDC, solved in a collaboration with W. Furey, Univ. of Pittsburgh), one can begin to assign function to individual amino acids at atomic resolution. During the current grant period methods were developed to prepare variants of PDC from the yeast Saccharomyces cerevisiae, using techniques in molecular biology. Yeast PDC is subject to regulation both by its substrate and by its cofactors thiamin diphosphate and Mg(II). During the current period two amino acids at the regulatory site have been assigned function (Cys221 and His92). These two amino acids are on different domains and it is hypothesized that when the first substrate binds to this locus, the information is transmitted to the catalytic site more than 20 Angstrom away. In addition, several amino acids in the catalytic center were identified as having a major impact not only on cofactor binding, but also on cofactor-induced regulation, as well as on catalysis. The goals for the coming period include the following outstanding problems: 1. delineation of the structural consequences of substitutions in the region responsible for activation by cofactors (the so-called thiamin diphosphate fold); 2. delineation of the entire substrate activation pathway; 3. delineation of the consequences of the unusual V coenzyme conformation; 4. delineation of the mechanism responsible for activation of the aminopyrimidine ring in catalysis; and 5. delineation of the function and chemical properties of potential general acid/base catalysts near the active center. Tools are being proposed, ranging from X-ray crystallography of wild-type and variant enzymes, to steady-state and pre-steady-state kinetics, to a variety of spectroscopic methods to help assign specific function for carrying out the chemical transformations in the mechanism to all of the amino acids implicated in regulation and catalysis. The PI believes that during the coming few years it will finally be possible to account for the 10E12-fold rate acceleration that the protein provides and to gain an intimate understanding of how enzyme-bound thiamin performs its function.
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