The overall objective of this research is to substantially extend our knowledge of structure-function relationships in a class of enzymes of fundamental importance to basic metabolism; the thiamin disphosphate dependent alpha-keto acid decarboxylases. Despite their widespread importance in biochemistry and many years of study, even the most basic structural information has only very recently become available for any member of this family, and numerous questions remain unanswered regarding the catalytic and regulatory mechanisms. The goal now is to exploit and expand upon the elementary structural information recently obtained for the representative enzyme pyruvate decarboxylase (PDC), by providing detailed information about structural changes resulting from interaction with allosteric activators, from inhibitors interacting at the catalytic and regulatory sites, and from single residue mutations introduced at important locations in both the regulatory and catalytic sites. This will be achieved by determining and analyzing the 3D structures of holoenzyme complexes and mutant enzymes via single crystal x-ray diffraction methods, and by comparing the structures with that for native PDC.
The specific aims are: (1) to determine and analyze the structure of PDC when complexed with the allosteric activators and substrate surrogates ketomalonate and pyruvamide; (2) to make detailed structural comparisons between complexes with activators and the native PDC structure, and to test several hypotheses regarding the allosteric activation mechanism; (3) to identify structural effects resulting from single residue mutations introduced in the catalytic site and which affect enzyme activity, and to test a hypothesis regarding cofactor stabilization by the enzyme; (4) to identify structural effects resulting from single residue mutations introduced in the regulatory site and which affect the allosteric activation process; (5) to examine the structure of the holoenzyme when frozen at intermediate stages along the reaction pathway by analysis of complexes with the catalytic site directed inhibitors glyoxalic acid and methyl acetylphosphinate; (6) to examine the structure of the holoenzyme after reaction with regulatory site directed inhibitors such as CPBs; (7) to make detailed structural comparisons between complexes with the inhibitors and the native PDC structure; (8) to obtain a detailed, high resolution structural analysis of the triclinic form of PDC.
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