Structural Studies of Thiamin Dependent Enzymes

Thiamin diphosphate (TDP, the vitamin B1 coenzyme) is one of the most important coenzymes in carbohydrate metabolism catalyzing e.g. the oxidative decarboxylation of pyruvate to acetylCoA in the reaction catalyzed by the pyruvate dehydrogenase multienzyme complex, enabling the product of glycolysis to enter the citric acid cycle. High resolution structural of such TDP-dependent pyruvate decarboxylating enzymes are being conducted by a variety of techniques. The principal target has been brewers'yeast pyruvate decarboxylase (PDC, E.C. 4 1.1.1) recently purified as fully active alpha4 and beta4 homotetramers in addition to the alpha2beta2 heteroteramer reporter earlier. In a most important and exciting recent development the alpha 4 structure was crystallized in the laboratory already providing novel information, and pointing to the likely success of resolution to at least 2.4 A. PDC is the most widely studied TDP-dependent eznyme and is therefore the best one on which to undertake the detailed structural studies proposed below. In addition to the x-ray developments major progress has been made in the spectroscopic characterization of enzyme-bound intermediates. It is proposed to extend these studies along the following lines of inquiry: 1. structural work on PDC by x-ray crystallographic methods (this part of the research is a collaborative effort with SAX, Fuery and coworkers at the VA Hospital Biocrystallography Lab/Univ. of Pittsburgh)n to determine: a. the structure of the 'TDP fold', identify the putative beta-turn-alpha-turn-beta structure at the TDP binding site; b. the basis of interaction between the TDP and Mg2+ with PDC; c./ the conformation of bound TDP, and of putative covalent intermediates; d. the nature of protein-protein interactions in PDC, i.e. among subunits in the absence and presence of the allosteric regulator pyruvamide; 2. mechanistic work of PDC, especially to elucidate the nonoxidative and oxidative chemistry of the enzyme-bound enamine; 3. immunochemical structural studies on PDC to determine immunochemical differences, if any, among isoforms; and on pyruvate dehydrogenase to probe its structure with the monoclones already at hand; 4. structural work on PDC and its TDP fold by NMR methods to determine the microenvironmental factors in catalysis. A collaboration has been initiated with S. Hohmann, who has cloned PDC in a variety of forms, and has and will supply the yeast containing the site-directed mutants, as well as the clones to address both structural and mechanistic questions.