Kinetics of Escherichia coli B D-Lactate Dehydrogenase and Evidence for Pyruvate-controlled Change in Conformation

Kinetic properties of d(-)-specific lactate dehydrogenase (d-lactate:diphosphopyridine nucleotide oxidoreductase, EC 1.1.1.28) from Escherichia coli B indicate the following. 1. The reaction catalyzed is essentially unidirectional, the oxidation of d(-)-lactate with diphosphopyridine nucleotide as coenzyme proceeding at only about 0.01% of the rate of pyruvate reduction. 2. The enzyme shows Michaelis-Menten kinetics for reduced diphosphopyridine nucleotide, but pyruvate activates it. 3. A lag in the rate of oxidation of the reduced diphosphopyridine nucleotide is eliminated by prior incubation with pyruvate. The duration of this lag is independent of enzyme concentration, suggesting that pyruvate causes a change in the conformation of the enzyme rather than in its state of aggregation. The identity of the molecular weight of the enzyme at a very low concentration in the absence and in the presence of the substrate confirms this finding. 4. Two lines of evidence indicate that there are two types of binding sites for pyruvate. The variation of Hill plots with pH is interpretable only in terms of two sites with different binding properties. Furthermore, the two sites bind different substrate analogues. Oxamate is an inhibitor of this enzyme, but does not substitute for pyruvate as an activator. On the other hand, α-ketobutyrate does not inhibit the catalysis but activates the enzyme, converting the substrate saturation curve from sigmoidal to hyperbolic. 5. The kinetic properties of the catalytic site appear to be similar to those of vertebrate l-lactate dehydrogenases, although the dissociation constants for both pyruvate and reduced diphosphopyridine nucleotide are considerably larger for the E. coli d-lactate dehydrogenase. 6. The kinetic properties of this enzyme are consistent with the course and regulation in vivo of pyruvate metabolism.