Lactic Dehydrogenase

The variation with pH of the kinetic parameters of the reaction catalyzed by bovine heart lactate dehydrogenase has been redetermined with use of purified coenzymes. Although some of the values differ from those reported previously by Winer and Schwert, the kinetic anomaly observed previously persists in the present results. This anomaly arises from the fact that the rate of dissociation of the enzyme-nicotinamide adenine dinucleotide complex, calculated on the assumption that the reaction follows a simple ordered pathway in which coenzymes must be bound before substrates can be bound, is less than the observed maximal velocity from the NADH-pyruvate side of the reversible reaction. This anomaly has been rationalized by assuming that at pH values below neutrality an unreactive protonated form of the enzyme-NAD complex is formed. Analysis of the results has permitted consistent values to be assigned to rate constants for every step in the reaction with the exception of the interconversion of the ternary enzyme-NAD-lactate and enzyme-NADH-pyruvate complexes. Silverstein and Boyer have concluded from measurements of the rate of interconversion of coenzymes and substrates at equilibrium that the reaction catalyzed by bovine heart lactate dehydrogenase follows a compulsory pathway at pH 7.9 but only a preferred order of combination with substrates and coenzymes at pH 9.7. An alternative explanation of the results obtained at pH 9.7 is proposed which is consistent with the reaction model deduced from kinetic parameters.