THE REGULATION OF ACETYL-COENZYME-A CARBOXYLASE IN RAT LIVER

LI-AN YEH, Purdue University

Abstract

The rate of phosphorylation and inactivation of rat liver acetyl-CoA carboxylase is controlled by ATP concentration. Maximum phosphorylation of the enzyme is attained in the presence of 0.4 mM ATP. Higher concentrations of ATP inhibit phosphorylation and inactivation of the carboxylase. Thus, in the presence of 4 mM ATP there is little or no phosphorylation of the enzyme. The addition of AMP stimulates phosphorylation and inactivation of acetyl-CoA carboxylase. In the presence of 1 mM ATP, the maximum stimulation of phosphorylation is obtained at about 2 mM AMP. The phosphorylation pattern of carboxylase examined at different adenylate energy charge levels generated by different concentrations of AMP and ATP indicates that phosphorylation and inactivation of the enzyme are regulated by the adenylate energy charge of the phosphorylation system. With an adenylate charge system containing 4 mM total adenylate, the maximum phosphorylation and inactivation occurred when the adenylate pool was made up of 1.6 mM ATP and 2.4 mM AMP. The effect of the adenylate energy charge on phosphorylation and inactivation of carboxylase suggest that inactivation of the enzyme due to phosphorylation is actually prevented in the presence of high levels of ATP and low AMP. This may explain how carboxylase can function in the presence of high ATP concentrations when fatty acid synthesis is expected to occur in vivo even though ATP is involved in the phosphorylation and inactivation of the enzyme. This findings also suggest that modest increases in AMP concentration will effectively shut off fatty acid synthesis by stimulating phosphorylation and inactivation of acetyl-CoA carboxylase. Acetyl-CoA carboxylase is activated by physiological concentrations of CoA. Activation of partially purified enzyme by CoA is accompanied by a decrease in the K(,m) for acetyl-CoA from 0.2 mM to about 4 (mu)M, which is the physiological concentration of acetyl-CoA in the cytosol. CoA activation of the purified enzyme is accompanied by an increase in the V(,max), without changing the K(,m) for acetyl-CoA. The k(,m) for acetyl-CoA of the purified enzyme is about 10 to 40 (mu)m. The purification procedure results in a decrease in the K(,m) for acetyl-CoA; under these conditions, CoA activation does not cause further lowering of the K(,m). CoA activation is accompanied by polymerization of the enzyme. However, CoA activation is not causally related to polymerization. There is one CoA binding site per subunit of acetyl-CoA carboxylase. CoA binding at that site is not affected by the presence of citrate, but palmityl CoA inhibits CoA binding. CoA alone cannot reverse palmityl CoA inhibition of the carboxylase. BSA and CoA together can activate the palmityl CoA inhibited enzyme. This indicates that the involvement of BSA-like protein, CoA, and palmityl CoA may play a physiologically significant role in the control of acetyl-CoA carboxylase. A method for the quantitation of CoA which can detect picomol quantities of coenzyme A is described. The method is based on coupling the enzymatic reactions of acetyl-CoA synthetase and acetyl-CoA carboxylase. This determination is suitable for tissue extracts with very low concentrations of CoA. Elimination of acetyl-CoA synthetase from the reaction mixture allows us to determine acetyl-CoA concentrations in biological samples. Further increases in the sensitivity of this method can be achieved by increasing the specific activity of KH('14)CO(,3).

Degree

Ph.D.

Subject Area

Biochemistry

COinS