Acetyl‐CoA‐Dependent Cleavage of Citrate on Inactivated Citrate Lyase

Abstract

Citrate lyase, an acetyl enzyme which catalyzes the cleavage of citrate in two consecutive steps, the acyl exchange and acyl lyase reactions, on deacetylation yields inactivated enzyme. On treatment with iodoacetate, this is irreversibly converted to another inactivated lyase. Both inactivated lyase species are catalytically active in the presence of certain acyl‐CoA derivatives such as acetyl‐CoA or citryl‐CoA. 3 S ‐Citryl‐CoA is cleaved stereospecifically to acetyl‐CoA and oxaloacetate in this catalysis, and citrate, in the presence of acetyl‐CoA, is cleaved to acetate and oxaloacetate. The acetyl group of acetyl‐CoA however is liberated as acetate and the pro‐S acetyl group of citrate is transformed to acetyl‐CoA during the acetyl‐CoA‐dependent cleavage of citrate. This exchange reaction was demonstrated by isotopic experiments. Kinetic analysis of the reaction indicated the formation of an intermediate. This conclusion was substantiated by performing the acetyl‐CoA‐dependent cleavage of citrate in the presence of EDTA. The complexing agent inhibits the lyase activity of the enzyme and therefore leads to the accumulation of the intermediate, 3 S ‐citryl‐CoA. The formation of 3 S ‐citryl‐CoA from acetyl‐CoA and citrate in the presence of EDTA demonstrates the acyl exchange activity, the cleavage of this intermediate demonstrates the lyase activity of inactivated citrate lyase. Comparison of these results with the mechanism of action of native citrate lyase led to the conclusion that the acetylated acyl carrier groups (enzyme‐ S ‐acetyl in citrate lyase; CoA‐ S ‐acetyl on inactivated citrate lyase) are alike in both systems. This was supported by the demonstration of the presence of phosphopantetheine in the lyase. From these and other results, citrate lyase was characterized as a multienzyme complex. It was concluded that phosphopantetheine most likely represents the acyl‐carrying group in this complex.