Liver Acetyl‐Coenzyme‐A Carboxylase

The mechanism of the liver acetyl‐coenzyme‐A carboxylase reaction was studied by kinetic experiments. Studies on inhibition by kynurenate, a novel dead‐end inhibitor binding to the uncarboxylated form of the enzyme, as well as malonyl‐CoA‐[ 14 C]acetyl‐CoA exchange studies further substaintiated our previous conclusions that the reaction proceeds through the ordered bi‐bi uni‐uni ping‐pong mechanism, and that the site of action of citrate, an allosteric activator, lies predominantly on the carboxylated form of the enzyme. The complex patterns produced by malonyl‐CoA inhibition were accounted for by mixed product and dead‐end inhibition; malonyl‐CoA was found to form a dead‐end complex with the inactive (or less active) species of the carboxylated form of the enzyme. On the other hand, the dissociation constant of the uncarboxylated enzyme · citrate complex was estimated to be 3–14 μM in terms of [ 14 C]citrate binding, protection against avidin inactivation and shift in the protomer‐polymer equilibrium. This value is about 10 3 ‐fold smaller than the dissociation constant of the carboxylated enzyme · citrate complex found by kinetic studies, i.e. 3–6 μM, so that the effect of citrate on the uncarboxylated form of the enzyme does not manifest itself upon kinetic analysis of the carboxylase‐catalyzed reactions. These findings indicate that carboxylation of the biotinyl prosthetic group gives rise to a conformational change at the citrate site, resulting in a marked decrease in the affinity of the enzyme for the activator. This further supports our previously proposed concept that the main role of citrate in the carboxylase catalysis is to keep the carboxylated form of the enzyme in active conformation by shifting the equilibrium between the active and inactive (or less active) species of this enzyme form.