Active oligomeric states of pyruvate decarboxylase and their functional characterization

Homomeric pyruvate decarboxylase (E.C 4.1.1.1) from yeast consists of dimer s and tetramers under physiological conditions, a K-d value of 8.1 mum was determined by analytical ultracentrifugation. Dimers and monomers of the en zyme could be populated by equilibrium denaturation using urea as denaturan t at defined concentrations and monitored by a combination of optical (fluo rescence and circular dichroism) and hydrodynamic methods (analytical ultra centrifugation). Dimers occur after treatment with 0.5 m urea, monomers with 2.0 m urea inde pendent of the protein concentration. The structured monomers are catalytic ally inactive. At even higher denaturant concentrations (6 m urea) the mono mers unfold. The contact sites of two monomers in forming a dimer as the sm allest enzymatically active unit are mainly determined by aromatic amino ac ids. Their interactions have been quantified both by structure-theoretical calculations on the basis of the X-ray crystallography structure, and exper imentally by binding of the fluorescent dye bis-ANS. The contact sites of t wo dimers in tetramer formation, however, are mainly determined by electros tatic interactions. Homomeric pyruvate decarboxylase (PDC) is activated by its substrate pyruva te. There was no difference in the steady-state activity (specific activity ) between dimers and tetramers. The activation kinetics of the two oligomer ic states, however, revealed differences in the dissociation constant of th e regulatory substrate (K-a) by one order of magnitude. The tetramer format ion is related to structural consequences of the interaction transfer in th e activation process causing an improved substrate utilization.