[pH-dependence of the structural and functional properties of lactate dehydrogenase (M4). Indirect cooperativity in lactate dehydrogenase].

The dependence of structural and functional properties of LDH on pH in the 6.0--9.0 region was investigated. There were no marked deviations of pyruvate reduction initial velocity curves from the Michaelis--Menten equation in a wide range of pyruvate concentrations. It was shown that Vmax changes negligibly in the 6.0--9.0 pH regions, but Km increased markedly with pH elevation. The pK value of 7.8+/-0.1 was obtained for 50% changes of pyruvate binding. The dependence of enzyme inhibition from pH at a high pyruvate concentration (20 mM) was investigated. At pH values above 8.0 pyruvate inhibition disappeared. The dependence of the inhibition degree from pH was estimated as pK 7.8+/-0.1. Hill coefficient (n) calculated from the curves of Km and the degree of substrate inhibition depending on pH was 1.6; n for pyruvate inhibition at pH 7.5 was 2 greater than n greater than 1 for moderate substrate concentrations (1--5 mM) and n approximately 1 for higher concentrations (5--40 mM). The value of n approximately 1 at pH 7.8 was obtained. The model suiting all available data concerning the cooperativity phenomena in LDH during protonation and inhibition by pyruvate is outlined. The model is based on the results indicating the slow isomerisation of LDH in ternary complexes with NADH and pyruvate and the absence of equilibrium on the intermediate stage of reaction.