MOLECULAR MECHANISM OF REGULATION OF THE PYRUVATE-DEHYDROGENASE COMPLEX FROM ESCHERICHIA-COLI

The pyruvate dehydrogenase multienzyme complex from E, coli shows a si gmoidal dependency of the reaction rate on the substrate concentration when product formation is followed in the presence of physiological c oncentrations of the cofactor thiamin diphosphate. To elucidate the mo lecular mechanism of this regulation, the influence of the substrate p yruvate on the coenzyme-protein interaction has been investigated usin g several coenzyme analogues. The observed binding constants of all co enzymatically active analogues are increased in the presence of the su bstrate pyruvate, whereas those of all coenzymatically inactive analog ues are not altered in the presence of pyruvate. This points to an inc reased binding affinity of a reaction-intermediate-coenzyme complex to the protein. Since cofactor binding and dissociation at physiological concentrations of thiamin diphosphate are slow compared to the cataly tic reaction, a slow transition to the active state of the enzyme occu rs. After lowering the pyruvate concentration! the opposite effect, a dissociation of the thiamin diphosphate from the enzyme is observed. T his slow substrate dependent enhancement of cofactor binding enables e fficient regulation of the pyruvate dehydrogenase complex by its subst rate pyruvate.