Kinetic Studies on the Mechanism of the Malate Dehydrogenase Reaction

This work is a kinetic investigation of the reaction mechanism of malate dehydrogenase, prepared from washed mince of whole bovine heart by a variation on previous methods. The forward and reverse reactions catalyzed by this enzyme have been studied at pH 8.0 in the presence and in the absence of one product at a time, with the use of a recording fluorometer to measure changes in the concentration of NADH. The initial velocity pattern in the absence of products and the product inhibition pattern have been determined. These are consistent with an ordered mechanism which has a kinetically significant ternary complex, and in which the coenzyme substrates combine with the free enzyme. Values have been determined for all the Michaelis, dissociation, and inhibition constants of the reaction. The dissociation constants determined for the coenzymes acting as substrates differ from estimates of the same constants obtained by studying the coenzymes as product inhibitors. These effects may be related to substrate inhibition by oxalacetate and substrate activation by malate, which have been observed previously. The rate constants calculated for the separate reaction steps in the mechanism reveal that the simplest Bi mechanism (Cleland, W. W., Biochim. Biophys. Acta, 67, 104 (1963)) does not apply; they are consistent with an Ordered mechanism in which the enzyme-oxidized coenzyme complex isomerizes. As with other dehydrogenases for which this condition applies, the possibility cannot be excluded that the enzyme-reduced coenzyme complex may also isomerize.