Studies on ATP Citrate Lyase of Rat Liver

The details of the reaction mechanism of ATP citrate lyase [EC 4. 1.3.8], especially on the mode of action of CoA, were investigated using a single homogeneous preparation from rat liver. The results obtained are: ( 1 ) The enzyme rapidly catalyzes the acetyl-CoA— CoA exchange, but not the acetyl-CoA— acetate exchange. ( 2 ) The combination of 14C-acetate to the protein occurs on incubation of 14C-acetyl-CoA with the enzyme, showing the formation of an enzyme-acetate complex. When this complex is isolated by gel filtration and then incubated with oxaloacetate, 14C-citrate is formed. The formation of an enzyme-oxaloacetate complex is not detected. (3 ) The enzyme easily combines with free CoA or CoA that is released from acetyl-CoA during the formation of the enzyme-acetate complex. This binding of CoA to the enzyme does not depend on the presence of added ATP, and the bound CoA is freely exchangeable with exogenous CoA. These data indicate that the linkage between enzyme and CoA is not energy-rich, unlike that between enzyme and acetate. From these findings, it is concluded that enzyme∼P,⋈COA, enzyme∼citrate⋈COA and enzyme∼acetate⋈COAare involved as intermediates in the reaction, and the possibility of an involvement of citryl-CoA as a reaction intermediate is excluded. An additional role of CoA, besides that of an acetyl acceptor, is also discussed.