FLEXIBLE ASSEMBLY OF PYRUVATE DECARBOXYLASE TETRAMERS EXAMINED BY COMPARISON OF MULTIPLE CRYSTAL FORMS. P. Arjunan, L. Chen, S. Swaminathan, W. Furey & M. Sax, Biocrystallography Laboratory, VA Medical Center, University Drive C, PO Box 12055, Pittsburgh, PA 15240 & Department of Crystallography, University of Pittsburgh, Pittsburgh, PA 15260 and Y. Gao, D. Zhang & F. Jordan, Department of Chemistry, Rutgers University, Newark, NJ 07102

Pyruvate decarboxylase (PDC) is a thiamin diphosphate (ThDP) dependent enzyme that catalyzes the non-oxidative conversion of pyruvate to acetaldehyde and carbon-dioxide. PDC is active as a tetramer of MW 250,000 KDa with four cofactor molecules (ThDP and Mg²⁺) tightly bound, and is hysteretically regulated by the substrate. It was shown by mutational analysis that Cys221 is required for the allosteric substrate activation, and low angle scattering studies indicate a change in tetramer assembly takes place upon activation. Kinetic studies also indicate that unlike the normal substrate pyruvate which results in reversible activation, the alternate substrate ketomalonate converts PDC to an activated form which remains "locked on" and does not undergo unimolecular inactivation.

Using wild type PDC from the yeast Saccharomyces uvarum and recombinant PDC from the yeast Saccharomyces cerevisiae, we have now determined the enzyme structure in five different crystal forms, two of which were obtained in the presence of the allosteric activator ketomalonate. The five crystal forms show three distinct variations of tetramer assembly, with the largest difference in one of the structures obtained in the presence of the irreversible activator. In all cases the major differences involve shifting of one dimer within the tetramer relative to the other, with the individual dimers remaining essentially intact. The central domain however, which is associated with regulation, shows significantly greater variation than either of the cofactor binding domains. Structural details and comparisons between the different crystal forms of PDC will be presented.