An L-(+)-lactate dehydrogenase was purified approximately 35-fold from crude extracts of Streptococcus faecalis. The purified enzyme had an absolute and specific requirement for fructose-1 ,6-diphosphate (FDP) for catalytic activity. The concentration of FDP required for 50% maximal activity was about 0.045 mm. The activator was bound to the enzyme more effectively at pH 5.8 than it was at a neutral or alkaline pH. Activation appeared to involve a conformational change in the enzyme which made the substrate and coenzyme sites more accessible to the respective reactants. Among the evidence supporting this hypothesis was the fact that FDP lowered significantly the apparent Km for both pyruvate and reduced nicotinamide adenine dinucleotide. Moreover, the enzyme, which was quite heat stable in the absence of any of the reactants, was rendered heat labile by FDP. 717 on July 16, 2020 by guest

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The lactate dehydrogenase (LDH) from Streptococcus mutans NCTC 10449 is under stringent metabolic control. The partially purified enzyme was specifically activated by high concentrations of fructose-1,6-diphosphate (FDP) and was inhibited by adenosine triphosphate. There appeared to be at least two binding sites for the activator which interacted in a cooperative manner. The interaction between the FDP sites was independent of the p H of the assay system, although the relative affinity of the enzyme for the activator was influenced by p H. There also appeared to be at least two pyruvate binding sites on the S. mutans LDH with some cooperative interaction between them, and the interaction between these sites was also independent of the hydrogen ion concentration. Two pyruvate analogues had different effects on the interaction of pyruvate with the LDH. One of the analogues, α-ketobutyrate, stimulated enzyme activity at limiting pyruvate concentrations, but had no significant effect at saturating concentrations of the substrate. The net effect of α-ketobutyrate was to shift the pyruvate saturation curve from sigmoidal to hyperbolic and to decrease the Hill coefficient from about 2.0 to 1.0. The other pyruvate analogue, oxamate, inhibited enzyme activity at all pyruvate concentrations but had no effect on the sigmoidal nature of the pyruvate saturation curve or on the apparent kinetic order of the reaction with respect to substrate. These results suggested that there may be two types of pyruvate binding sites on the LDH from S. mutans . Other kinetic properties of the S. mutans NCTC 10449 enzyme were studied and compared with those exhibited by the LDH from several other strains of the organism.