Studies on the Mechanism of Activation of Acetyl Coenzyme A Carboxylase by Citrate

Abstract

The observation by Brady and Gurin (1) that certain tricarboxylic acid cycle intermediates stimulate fatty acid synthesis has been confirmed in many laboratories (2-9). Until recently, however, the mechanism of this stimulation has remained obscure. The discovery by Wakil of acetyl coenzyme A carboxylase (lo), the biotin enzyme that catalyzes the adenosine triphosphate-dependent carboxylation of acetyl coenzyme A to form malonyl coenzyme A, and the involvement of malonyl coenzyme A in the biosynthesis of fatty acids, led to intensive studies of possible control mechanisms in the regulation of fatty acid synthesis. Ganguly (11) and IVuma, Matsuhashi, and Lynen (12) reported that carboxylation of acetyl coenzyme A is the rate-limiting reaction in fatty acid biosynthesis. It has been reported recently that tricarboxylic acid cycle intermediates, most notably citrate, stimulate fatty acid synthesis in an enzyme system from rat adipose tissue through the activation of acetyl-CoA carboxylase (13-15). Similar conclusions have been drawn independently from experiments in an avian liver preparation (16) and in a rat liver preparation (17). Evidence has been found in the enzyme system from adipose tissue (15) that citrate did not take part in the reaction by providing COZ by a direct transcarboxylation mechanism. In addition, prior incubation of the enzyme with citrate alone caused activation that could not be explained by the formation of a free intermediate from citrate which was responsible for activation. It was therefore suggested that the carboxylase itself is activated during the prior incubation with citrate. This paper presents the results of further studies on the mechanism of activation of acetyl-CoA carboxylase by citrate in the adipose enzyme system. The possibility that citrate activates the enzyme by virtue of its simple chelating activity is ruled out by comparison with a series of effective chelating agents. Experiments with trans-aconitate and fluorocitrate, two potent inhibitors of the Krebs cycle, rule out the possibility that citrate activates the carboxylase by giving rise to catalytic amounts of Krebs cycle intermediates. Evidence is presented that citrate activation of the carboxylase is associated with alteration of the sedimentation characteristics of the enzyme. A preliminary report of this work has appeared (18).