ATP‐citrate lyase from rat liver

The mechanism of ATP‐citrate lyase has been proposed to involve a citryl‐enzyme intermediate. When the enzyme is incubated with its substrates ATP and [ 14 C]citrate, but in the absence of the final acceptor, two distinct types of citrate‐containing complex can be isolated. At early time points, a highly unstable complex can be isolated by gel filtration which has a half‐life of 36 s at 25°C. This complex reacts rapidly with CoA, but cannot be acid‐precipitated; behaviour consistent with its identification as enzyme‐citryl phosphate. However, ATP‐citrate lyase is also capable of undergoing a slow time‐dependent covalent incorporation of radiolabel from [ 14 C]citrate. This modification is acid‐stable, non‐specific, and cannot be reversed by the addition of CoA. When cytochrome c is included in the reaction mixture as a heterologous acceptor, it is also citrylated. These reactions require that when ATP‐citrate lyase is incubated with all its substrates except for CoA, a freely diffusible citrylating species is generated within the active site. This evidence suggests that there is no requirement for the mechanism of ATP‐citrate lyase to proceed via a covalent citryl‐enzyme intermediate. By analogy with succinyl‐CoA synthetase, an enzyme which has a high degree of sequence similarity with ATP‐citrate lyase, a simple mechanism is proposed for the enzyme in which citryl‐CoA is produced by direct nucleophilic attack on citryl phosphate.