Liver Acetyl Coenzyme A Carboxylase

Abstract The factors effecting the reversible interconversion between the protomeric and polymeric forms of liver acetyl coenzyme A carboxylase were investigated by sucrose density gradient centrifugation and electron microscopy. Certain anions (citrate, isocitrate, malonate, tricarballylate, sulfate, and Pi), acetyl-CoA, high protein concentration, and pH 6 to 7 promote aggregation of the protomer. Other factors, such as carboxylation of the enzyme to produce enzyme-CO2-, Cl-, and pH values greater than 7.5, cause dissociation of the polymeric form. Citrate and isocitrate, but not tricarballylate or Pi, are capable of promoting the transition of the carboxylated enzyme (enzyme-CO2-) to the polymeric state. Under the conditions for carboxylase assay or for measuring the rates of other carboxylase-catalyzed reactions, all of which involve enzyme-CO2- as intermediate, isocitrate and citrate promote aggregation of the carboxylase to its polymeric form and a 15- to 16-fold activation of catalysis; neither tricarballylate nor Pi has this effect. Binding experiments reveal that acetyl-CoA carboxylase has one tight binding site per protomer (mol wt 410,000) for citrate (Kd, 2 to 3 x 10-6 m) and another for acetyl-CoA (Kd, 4 x 10-6 m). Acetyl-CoA binding at the latter site is unaffected by the presence of citrate. Dissociation of acetyl-CoA carboxylase with sodium dodecyl sulfate (SDS; 0.1%) gives rise to subunits with a sedimentation coefficient (s20,w) of 4.3 S. By equilibrium dialysis against 0.1% SDS-35S, the carboxylase was found to bind 0.57 g of the detergent per g of carboxylase protein. The subunit molecular weight, determined by sedimentation equilibrium corrected for detergent binding, was 114,000 and by electrophoresis in SDS-polyacrylamide gels (1% SDS), 110,000. The presence of nonidentical subunits is indicated by the fact that there is a single biotinyl prosthetic group and there are single binding sites for both citrate and acetyl-CoA on the 410,000 molecular weight protomer.