Pyruvate carboxylase. Studies of activator-independent catalysis and of the specificity of activation by acyl derivatives of coenzyme A for the enzyme from rat liver.

Abstract Oxalacetate synthesis catalyzed by pyruvate carboxylase from rat liver in the absence of acetyl-CoA exhibits a pH dependence and specificity for activation by univalent and divalent cations similar to that reported previously for acetyl-CoA-dependent oxalacetate synthesis by this enzyme (Mc-Clure, W. R., Lardy, H. A., and Kneifel, H. P. (1971) J. Biol. Chem. 246, 3569–3578). Fractionation studies have provided no indication that different species are responsible for catalysis in the presence or absence of this activator. However, linear Arrhenius and van't Hoff plots are observed for the temperature dependence of oxalacetate synthesis in the absence of acetyl-CoA over the range 10–45° and Ea is obtained as 15.4 Cal per mole. In contrast in the presence of acetyl-CoA biphasic Arrhenius and van't Hoff plots are observed over this temperature range with the changes in slope occurring at approximately 25°. The values obtained for Ea above and below 25° are 9.1 and 31.5 Cal per mole, respectively. Hence the extent of activation of the maximal rate of oxalacetate synthesis by acetyl-CoA is a function of the temperature of observation. The apparent Ka describing activation of rat liver pyruvate carboxylase by acetyl-CoA is a function of [pyruvate] but shows no significant dependence on [MgATP2-] or [HCO3-]. The apparent Ka decreases from a value of 145 µm at [pyruvate] → 0 to 50 to 55 µm as the pyruvate concentration is increased to saturation. These data, which may be relevant to in vivo regulation of the pyruvate → oxalacetate flux, indicate a specific interaction between the catalytic and activator sites of rat liver pyruvate carboxylase. Examination of the specificity of activation of rat liver pyruvate carboxylase by acyl derivatives of coenzyme A and related compounds has shown that acetyl-CoA is the most potent activator of this enzyme. Effective activation is also observed in the presence of alkylacyl homologs in which the acyl chain contains 3 (propionyl-CoA) to 12 (n-dodecanoyl-CoA) carbon atoms while other derivatives, e.g. CoA-SH, adenosine 3':5'-diphosphate, and phenylacetyl-CoA, are weak activators. Carboxyacyl derivatives of coenzyme A, e.g. succinyl-CoA, and derivatives in which the phosphoadenosyl moiety is modified (e.g. acetyl-3'-dephospho-CoA, acetyldeamino-CoA, adenosine 2':5'-diphosphate) act as inhibitors of rat liver pyruvate carboxylase. The Hill coefficient describing the relationship between initial rate and activator concentration approximates 2.0 for all activators except adenosine 3':5'-diphosphate indicating that the presence of the 4'-phosphopantetheine portion of the CoA molecule is necessary for observation of cooperative interaction between activator sites. The presence of an alkylacyl moiety enhances the effectiveness of activation as indicated by an increased Vmax and, in some instances, a more favorable apparent Ka. Since longer chain alkylacyl derivatives of coenzyme A act as inhibitors of chicken liver pyruvate carboxylase these data also indicate a marked difference in the response of the chicken and rat liver enzymes to the size of the acyl moiety.