Multiple interaction of fructose 1,6-bisphosphate and other effectors on phosphoenolpyruvate carboxylase from Brevibacterium flavum and its aspartate-producing mutant.

Abstract

Phosphoenolpyruvate (PEP) carboxylase purified from Brevibacterium flavum was specifically activated by fructose 1, 6-bisphosphate (FBP). The other intermediates of sugar metabolism or their structural analogues did not influence the activity. FBP decreased the apparent Km for PEP but did not affect that for another substrate, bicarbonate, or the apparent maximum velocity for PEP. The dissociation constants for FBP from enzyme-FBP and enzyme-PEP-FBP complex were 63 and 32 μM, respectively, being almost equivalent to those for acetyl-CoA. Synergistic activation by FBP and acetyl-CoA was not observed with the B. flavum enzyme, unlike the Escherichia coli enzyme. FBP, like acetyl-CoA, was kinetically competitive with aspartate. With respect to another feedback inhibitor, 2-oxoglutarate, acetyl-CoA was non-competitive, whereas FBP was of mixed-type, i.e., FBP but not acetyl-CoA prevented 2-oxoglutarate from binding to the enzyme to a certain extent. Homotropic cooperativity was observed only with FBP but not with acetyl-CoA in the absence of inhibitors. Cooperativities of FBP and acetyl-CoA were increased by aspartate but not by 2-oxoglutarate. In the aspartate-overproducing mutant enzyme, the Michaelis constant for PEP was decreased, whereas the inhibitor constant for aspartate with or without simultaneous addition of 2-oxoglutarate and the activator constants for FBP and acetyl-CoA were increased. The decreased Michaelis constant for PEP was comparable to the apparent Km of the wild-type enzyme for PEP in the presence of the saturated concentration of FBP, and would result in a further decrease in the affinity of the mutant enzyme for aspartate.