Studies on the regulation of L-aspartate β-decarboxylase of Alcaligenes faecalis were carried out under conditions in which aspartate is decarboxylated to α-alanine at a constant rate. Addition of α-keto acid increases the Vmax value for decarboxylation about sixfold at 37°, but the Km values for aspartate in the presence and absence of added α-keto acid are about the same (about 6 × 10-4 m). When the enzyme is incubated with aspartate (in absence of added α-keto acid) there is an initial rapid decarboxylation associated with a burst of pyruvate formation followed by slower but linear decarboxylation. There is no net increase in pyruvate concentration during the latter phase. Studies in which lactate and malate dehydrogenases and reduced diphosphopyridine nucleotide were added indicate that pyruvate is formed by decarboxylation and hydrolysis of the ketimine Schiff base intermediate and that pyruvate is essential for continued decarboxylase activity. A constant rate of decarboxylation results from an equilibrium between enzyme-bound alanine-ketimine and enzyme-bound pyridoxamine 5′-phosphate and pyruvate. The enzyme can bind close to 1 mole of α-keto acid/minimal catalytic unit; studies with the inhibitory aspartate analog β-cyanoalanine (Ki = 2.7 × 10-4 m) indicate that the α-keto acid binding site is different from that for aspartate. The apoenzyme can also bind pyruvate effectively. The activity of the enzyme is increased by adding very low concentrations of pyruvate and decreased by removal of pyruvate; thus, the enzyme would be subject to highly sensitive regulation in an intracellular environment containing other enzymes capable of increasing or decreasing the concentrations of pyruvate, α-ketoglutarate, and other α-keto acids. This allosteric regulatory mechanism is novel in that the α-keto acid effector is produced by the enzyme from the substrate; the effector has no effect on substrate affinity, and reacts with the inactive enzyme-bound coenzyme to yield active coenzyme. © 1969, American Chemical Society. All rights reserved.
