L-aspartate is a regulatory feedback inhibitor of the biotin-dependent enzyme pyruvate

carboxylase in response to increased levels of tricarboxylic acid cycle intermediates. Detailed

studies on L-aspartate inhibition of pyruvate carboxylase have been mainly confined to eukaryotic

microbial enzymes and aspects of its mode of action remain unclear. Here we examine its

inhibition of the bacterial enzyme Rhizobium etli pyruvate carboxylase. Kinetic studies

demonstrated that L-aspartate binds to the enzyme cooperatively and inhibits the enzyme

competitively with respect to acetyl CoA. L-aspartate also inhibits activation of the enzyme by

MgTNP-ATP. The action of L-aspartate was not confined to inhibition of acetyl CoA binding,

since the acetyl CoA-independent activity of the enzyme was also inhibited by increasing

concentrations of L-aspartate. This inhibition of acetyl CoA-independent activity was

demonstrated to be focused in the biotin carboxylation domain of the enzyme and it had no effect

on the oxamate-induced oxaloacetate decarboxylation reaction that occurs in the carboxyl

transferase domain. L-aspartate was shown to competitively inhibit bicarbonate-dependent

MgATP cleavage with respect to MgATP, but also probably inhibits carboxybiotin formation

and/or translocation of the carboxybiotin to the site of pyruvate carboxylation. Unlike acetyl CoA,

L-aspartate has no effect on the coupling between MgATP cleavage and oxaloacetate formation.

The results suggest that the three allosteric effector sites (acetyl CoA, MgTNP-ATP and L-aspartate)

are spatially distinct but are connected by a network of allosteric interactions.