Pyruvate Carboxylase from Chicken Liver

The interaction of pyruvate carboxylase purified from chicken liver with univalent and divalent metal ion activators has been investigated by a steady state kinetic analysis and also by examination of the effect of these activators on the rate of inactivation observed on incubation at 2°. Previous studies (Barden, R. E., Fung, C.-H., Utter, M. F., and Scrutton, M. C. (1972) J. Biol. Chem. 247, 1323) have shown that each active site on this enzyme is composed of two functionally distinct catalytic subsites which are linked by a biotinyl residue which serves as carboxyl carrier. In the present studies the kinetic analysis indicates that the divalent metal ion activator (Mg2+) interacts only at the subsite which catalyzes the carboxylation of the biotinyl residue. This observation is consistent with the results of previously reported isotope exchange studies (Scrutton, M. C., Keech, D. B., and Utter, M. F. (1965) J. Biol. Chem. 240, 574). In contrast, the univalent metal ion activator (K+) appears to interact at both the carboxylation subsite and also the subsite which catalyzes the transfer of the carboxyl group from the carboxylated biotinyl residue to pyruvate. The binding of MgATP2- to the enzyme strongly enhances the binding of Mg2+, and vice versa; a similar relationship exists between K+ and HCO3-. In the latter case the kinetic data indicate an equilibrium-ordered mechanism of addition with HCO3- adding first. Interaction of the enzyme with K+ markedly enhances the interaction with Mg2+, and vice versa. However, only a minimal enhancement of interactions is observed in the case of the K+-acetyl coenzyme A pair. When Mg2+ and acetyl-CoA are varied as an activator-activator pair, the initial rate versus [Mg2+] profile exhibits a significant extent of positive cooperativity in the presence of nonsaturating concentrations of acetyl-CoA but approaches classical behavior as acetyl-CoA is increased to saturation. Variation of Mg2+ does not, however, alter the extent of positive cooperativity which characterizes activation by acetyl-CoA. When catalytic activity is measured in the presence of various alkylammonium cations, the data obtained suggest that pyruvate carboxylase from chicken liver may catalyze a small but significant rate of oxalacetate synthesis in the absence of an added activating univalent cation. K+, NH4+, Rb+, Cs+, and T1+ are effective activators of this enzyme. Other univalent cations act either as weak activators (Tris+), or as noncompetitive inhibitors with respect to activation by K+ (Li+, Na+).