Abstract
We investigated the allosteric and kinetic properties of the heat-stable L-lactate dehydrogenase (EC 1.1.1.27) from Thermus caldophilus GK24. In the absence of an effector, the pyruvate and L-lactate saturation curves for the enzyme were sigmoid. In the presence of 0.2 mM fructose 1, 6-bisphosphate, the most potent effector, the substrate saturation curves ceased to show the positive cooperativities. The maximum velocity of pyruvate reduction increased 7-fold on the addition of fructose 1, 6-bisphosphate. This fructose 1, 6-bisphosphate-dependent enzyme activity was strongly inhibited by high concentrations of the substrate (pyruvate). In contrast, the NADH and NAD+ saturation curves for the enzyme were hyperbolic independent of fructose 1, 6-bisphosphate. Glucose 1, 6-bisphosphate, fructose 2, 6-bisphosphate and 5-phosphoribosyl 1-pyrophosphate also activated the enzyme to some extent. Inorganic phosphate slightly activated the enzyme, while it was rather inhibitory for the fructose 1, 6-bisphosphate-dependent enzyme reaction. Oxamate, an analogue of pyruvate, activated the enzyme and caused the positive cooperativity for pyruvate to disappear. The intracellular concentrations of fructose 1, 6-bisphosphate and pyruvate and the kinetic properties of L-lactate dehydrogenase of T. caldophilus Gk24 indicate that the enzyme exhibits the pyruvate reduction activity in concert with glycolysis under allosteric regulation by fructose 1, 6-bisphosphate.
