Kinetic Studies on the Inhibition of a d(-)-Specific Lactate Dehydrogenase by Adenosine Triphosphate

Abstract

A purified, NADH-linked, d (-)-specific lactate dehydrogenase from Butyribacterium rettgeri was shown to be inhibited by ATP. Several other nucleotides were also capable of inhibiting the reaction catalyzed by this enzyme but, at equivalent concentrations, none was found to be as effective as ATP. The inhibition by ATP appears to be of the partially competitive type with respect to NADH. This suggests that ATP may interact with the enzyme at a site distinct from the coenzyme-binding site. On the other hand, ATP seems to be an uncompetitive inhibitor with respect to pyruvate, i.e. at any given concentration of ATP, maximum inhibition was observed only when the enzyme was fully saturated with pyruvate. Although pyruvate appeared to increase the affinity of the lactate dehydrogenase for ATP, it was shown to cause a slight increase in the apparent K m for NADH. This differential effect of the substrate on the apparent affinities of the enzyme for the inhibitor and its coenzyme supports the hypothesis that separate sites are involved in binding the two compounds. Interactions of the enzyme with its coenzyme or the nucleotide inhibitor may result in conformational alterations of the protein. ATP protected the enzyme against heat inactivation and NADH reversed this protective effect, although the coenzyme itself had no effect on the heat lability of the enzyme.