[Kinetic studies of the formation of abortive ternary complex lactate dehydrogenase (isoenzyme h4)-NAD-pyruvate]

Abstract

The rate constants and equilibrium constants for four stages of process of the abortive ternary complex lactate dehydrogenase (porcine isoenzyme H4)-NAD-pyruvate formation are determined. These stages are 1) the enolization of pyruvate, 2) the formation of binary complex enzyme-NAD, 3) the formation of "intermediate" ternary complex enzyme-NAD-enol and 4) the transformation of "intermediate" complex into "final" ternary one, accumulation of the latter being followed by spectrophotometric and fluorometric methods. The constants obtained were compared with the corresponding ones for porcine isoenzyme M4 determined in earlier work. It was shown that the greater stability of ternary complex and the greater initial rate of ternary complex formation in the case of H4 is due to greater affinity of isoenzyme H4 with respect to NAD, greater magnitude of reaction rate constant for transformation of "intermediate" ternary complex into "final" one and lesser magnitude of reaction rate constant for the reverse transition.