Pyruvate dehydrogenase component of the pyruvate dehydrogenase complex from Escherichia coli K12. Purification and characterization.

Free pyruvate dehydrogenase component of the Escherichia coli K12 pyruvate dehydrogenase complex was isolated from a mutant lacking the dihydrolipoamide transacetylase component. The procedure, employing three chromatographic steps, yields a product that is electrophoretically pure. The purified enzyme reassociates with the residual complex lacking this component to a fully active enzyme complex. The kinetic characteristics of the free component were compared to that of the enzyme integrated into the native complex molecule. No essential differences could be detected regarding the behaviour of the catalytic reaction with variations in temperature, pH and substrate concentration. An inhibition, competitive to pyruvate, of the pyruvate dehydrogenase component (Ki = 18 microM) by fluoropyruvate was observed with both enzyme forms. Pyruvate exerts a cooperative effect both upon the partial enzyme reaction of the pyruvate dehydrogenase component as well as upon the overall reaction of the native enzyme complex. However, there is a clear difference in the shape of the saturation curves of both types of reaction. Experiments with the free enzyme, with the component integrated into the native complex molecule and with enzyme complexes which are partially deficient in the pyruvate dehydrogenase component demonstrate that the type of saturation curves obtained were characteristic for the reaction observed rather than for the interaction of a high number of subunits of the pyruvate dehydrogenase complex.