Pyruvate Carboxylase

Various intermediates of the tricarboxylic acid cycle and related compounds have been examined as inhibitors of pyruvate carboxylase purified from chicken and rat liver. These studies have indicated that α-ketoglutarate is a specific inhibitor of chicken liver pyruvate carboxylase. Addition of α-ketoglutarate causes the apparent Ka for acetyl coenzyme A to become less favorable but does not affect the apparent Vmax. However, magnetic resonance, cold inactivation, and group-specific modification studies indicate that this kinetic interaction is not the result of competition for the same binding site(s). The initial rate-[α-ketoglutarate] profile becomes increasingly more sigmoidal as [acetyl-CoA] is inincreased toward saturation. At [acetyl-CoA] → 0 the apparent Ki and Hill coefficient for α-ketoglutarate are obtained as 3.0 and 1.5 mm, respectively. α-Ketoglutarate acts as a noncompetitive inhibitor when MgATP2- or pyruvate is the varied substrate. Secondary plots of slopes or intercepts are nonlinear functions of [α-ketoglutarate]. When HCO3- is the varied substrate, addition of α-ketoglutarate induces non-linearity in the relationship between reciprocal initial rate and reciprocal [HCO3-]. In the presence of α-ketoglutarate a linear asymptote which is observed in the double reciprocal plots at [HCO3-] below 5 mm extrapolates to a negative intercept on the ordinate axis, suggesting that the addition of this inhibitor causes the HCO3- sites on the enzyme to become kinetically nonidentical. In contrast, l-glutamate is approximately equally effective as a classical inhibitor of pyruvate carboxylase purified from chicken and rat liver. Inhibition of both enzymes is uncompetitive with respect to MgATP2- and noncompetitive with respect to HCO3-. When acetyl-CoA is the variable component, addition of l-glutamate causes the apparent Ka for the activator to become less favorable and also induces a small but significant decrease in the apparent Vmax. With pyruvate as the varied substrate, l-glutamate acts as a simple competitive inhibitor of chicken liver pyruvate carboxylase. Inhibition of the rat liver enzyme is a complex function of [pyruvate] but approaches competitive behavior at high concentrations of this substrate. The apparent Ki for l-glutamate obtained from these data approximates 4 mm for both chicken and rat liver pyruvate carboxylases. Interaction of α-ketoglutarate and l-glutamate at different sites on chicken liver pyruvate carboxylase is suggested both by the marked dissimilarities between the properties of inhibition by these two metabolites and by studies which demonstrate the absence of kinetic interaction between the two inhibitors. It is suggested that the chicken liver enzyme carries specific regulatory sites at which α-ketoglutarate interacts, while the inhibition patterns observed for l-glutamate appear generally consistent with the proposal that this metabolite interacts at the α-keto acid site when the enzyme is in the form E-biotin ∼ CO2. Regulation of the catalytic activity of chicken liver pyruvate carboxylase by the α-ketoglutarate to l-glutamate ratio has also been demonstrated under conditions which may approximate some parameters of the in vivo environment.