NAD-linked L(+)-lactate dehydrogenase from the strict aerobe alcaligenes eutrophus. 2. Kinetic properties and inhibition by oxaloacetate.
The L(+)-lactate dehydrogenase (EC 1.1.1.27) of Alcaligenes eutrophus catalyzes the NADH-dependent reduction of pyruvate and a few other 2-oxoacids. The Km values for NADH, NAD, pyruvate and L(+)-lactate are 0.075 mM, 0.130 mM, 0.820 mM and 7.10 mM, respectively. The reaction follows a rapid equilibrium ordered bi-bi mechanism and involves the formation of a dead-end EBQ complex. The competitive inhibition of pyruvate reduction caused by NAD (with respect to NADH) is regarded to be of physiological importance. The enzyme is strongly inhibited by oxaloacetate, oxalate and to a less extent by oxamate. Oxaloacetate was found to be the most powerful inhibitor of the enzyme and exerts an almost complete inhibition of the reduction of pyruvate and some 2-oxoacids at concentrations of 1 microM and less. At 0.1 microM oxaloacetate the inhibition of pyruvate reduction is about 90%. The kinetics of pyruvate reduction in the presence of oxaloacetate is characterized by a burst phase followed by a decreased steady-state velocity. During the burst phase, which lasts from several seconds to some minutes, the enzyme undergoes transition to a less active enzyme form. The inhibition studies revealed the lactate dehydrogenase to be a hysteretic enzyme, due to its slow response to the ligand. The characteristics of the transient were examined. The inhibition of lactate dehydrogenase from A. eutrophus by oxaloacetate is considered to be of great physiological importance, allowing its function only at a low oxaloacetate concentration and consequently at high NADH/NAD ratios.
D000421:Alcaligenes / Q000201:enzymology*; D055598:Chemical Phenomena; D002621:Chemistry; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D009243:NAD / Q000378:metabolism; D010071:Oxaloacetates / Q000494:pharmacology*
D010071:Oxaloacetates; D009243:NAD; D007770:L-Lactate Dehydrogenase