Synergistic inhibition of phosphoenolpyruvate carboxylase by aspartate and 2-oxoglutarate in Brevibacterium flavum.

Purification procedures for phosphoenolpyruvate carboxylase from B. flavum were improved by using hydrophobic chromatography. The carboxylase showed optimum pH values of 7.2 and 8.0 with Mn2+ and Mg2+ as metallic activators, respectively. Purified phosphoenolpyruvate carboxylase was found to be synergistically inhibited by aspartate and 2-oxoglutarate in the absence or presence of an activator, acetyl-CoA. Similarly to the aspartate inhibition, 2-oxoglutarate alone inhibited the enzyme competitively with respect to both substrates, with an inhibitor constant of 4.7 mM. The dissociation constant for the combination of enzyme-2-oxoglutarate (-aspartate) complex with aspartate (2-oxoglutarate) was found to be one-third of that for the combination of the enzyme with aspartate (2-oxoglutarate). The Hill coefficient for phosphoenolpyruvate was increased from 1.0 to 2.3 by the simultaneous addition of the two inhibitors in a certain concentration range of phosphoenolpyruvate where strong synergistic effects were observed. Outside this concentration range, the coefficient was not altered or was slightly increased by the addition of aspartate, 2-oxoglutarate, or both. The synergistic action seems to be caused by these effects, in addition to the decrease in dissociation constants of the inhibitors. Hill coefficients for aspartate and 2-oxoglutarate were both approximately 2.0. The coefficient for one inhibitor did not vary with the addition of the other inhibitor. Although many structural analogues of the two inhibitors, such as 2-oxoadipate and 3-hydroxyaspartate, were very weak inhibitors, their synergistic effects with aspartate or 2-oxoglutarate were comparable to the effects of the two natural inhibitors. On the other hand, malate and succinate, which markedly inhibited the enzyme, did not show synergistic action with aspartate or 2-oxoglutarate. Hill coefficients for the structural analogues showing synergistic effects were approximately 2.0 or above, whereas those for malate and succinate, which did not enhance the inhibitions, were about 1.0. Phosphoenolpyruvate carboxylase from an aspartate-producing mutant had the inhibitor constant of 5.8 mM for 2-oxoglutarate, i.e., slightly higher than wild-type enzyme. The inhibitor constant for aspartate was three times higher than that of the wild-type enzyme as reported previously. The dissociation constant for aspartate of the enzyme-aspartate-2-oxoglutarate complex in the mutant enzyme was 8 times that in the wild-type enzyme, indicating that weaker synergistic inhibition was observed with the mutant enzyme.