Activation of rabbit muscle lactate dehydrogenase by phosphate: Active enzyme gel chromatography and enzyme kinetic studies

Abstract

Enzyme kinetic studies are presented which demonstrate the activating effect of phosphate on the conversion of pyruvate to lactate by rabbit muscle lactate dehydrogenase. A simple method of active enzyme gel chromatography is used to preclude the possibility that this effect is due to redistribution of enzyme between tetrameric and dissociated states as the result of preferential binding of phosphate to the tetrameric enzymatic form. By analysis of the kinetic results in terms of an ordered two-substrate mechanism, the source of the activation is traced to enhancement of the strength of the enzyme-NADH interaction, primarily because of an increase in the rate constant for the formation of the binary enzyme-coenzyme complex. Preliminary estimates of the relevant equilibrium constants from the kinetic data indicate that the binding of phosphate to rabbit muscle lactate dehydrogenase leads to a two- to fourfold increase in the intrinsic association constant for the interaction between NADH and the enzyme under the conditions (pH 7.4, I = 0.15) used to study the activation phenomenon.