Sheep kidney pyruvate carboxylase. Studies on its activation by acetyl coenzyme A and characteristics of its acetyl coenzyme A independent reaction.

The carboxylation of pyruvate in the absence of acetylCoA by sheep kidney pyruvate carboxylase has been demonstrated and the characteristics of the reaction have been investigated. Under optimal conditions, the maximal specific activity for the reaction was 25 % of that obtained in the presence of saturating concentrations of acetyl-CoA. The reaction was markedly stimulated by K+, NH4+, Rb+, and Csi-, particularly in the presence of sulfate ions. The specific activity of the enzyme was shown to be dependent upon the enzyme concentration in the assay solution; this was due to a concentration-dependent inactivation of the enzyme on dilution at pH 8.4 in the absence of acetyl-CoA. The addition of acetyl-CoA protected against inactivation on dilution and increased the catalytic activity of the enzyme. Acetyl-CoA induced a substantial decrease in the apparent K, values for bicarbonate and pyruvate without affecting the apparent K, value for MgATP2-. In the presence of acetylCoA the enzyme was less dependent on monovalent cations for activity, the apparent K, values for activating cations were lower and the cation specificity was altered. After taking these factors into account, it was apparent that the activator also greatly increased Vlnax. Pyruvate carboxylases isolated from the livers of rats, chickens, and sheep were also shown to catalyze the fixation of HnC03in the absence of acetyl-CoA.