ATP-citrate lyase from rat liver. Characterisation of the citryl-enzyme complexes.
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 [14C]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 degrees 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 [14C]citrate. This modification is acid-stable, non-specific, and cannot be reversed by the addition of CoA. When cytochrome 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.
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Molecular Enzymology Laboratory, SmithKline Beecham PLC, Welwyn, England.
D001275:ATP Citrate (pro-S)-Lyase / Q000378:metabolism*; D000818:Animals; D002850:Chromatography, Gel; D003065:Coenzyme A / Q000378:metabolism; D003574:Cytochrome c Group / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008297:Male; D010750:Phosphoproteins / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains
D003574:Cytochrome c Group; D010750:Phosphoproteins; D001275:ATP Citrate (pro-S)-Lyase; D003065:Coenzyme A