The long term goal of this proposal is the elucidation of the structure and mechanism of action of thiamin diphosphate- dependent pyruvate decarboxylating enzymes; pyruvate decarboxylase and pyruvate dehydrogenase complex. The latter enzyme is at a key junction in metabolism; a thorough understanding of its structure and mechanism is of fundamental biochemical importance. The enzymes will be purified in part by immunoaffinity chromatographic methods. The specific objectives are to: 1. design and synthesize mechanism-based inhibitors identify the sequence of the coenzyme and substrate binding sites and determine the location (including surrounding sequence) of the key Cys SH's of the enzymes. 2. determine the structure of the key enzyme-bound enamine using electronic spectroscopy. Ascertain its reactivity vis-a-vis protonation, disulfides, and flavin, in reactions mimicking all the important classes of thiamin diphosphate-dependent alpha-keto acid decarboxylating enzymes.
