Pyruvate Carboxylase

Incubation of pyruvate carboxylase purified from rat liver or Saccharomyces cerevisiae with 0.5 mm trinitrobenzene sulfonate at pH 7.2 causes rapid inactivation of acetyl coenzyme A-dependent oxalacetate synthesis with little concomitant decrease in (a) the rate of oxalacetate synthesis which occurs in the absence of acetyl-CoA or (b) (for pyruvate carboxylase from Saccharomyces cerevisiae) the extent of inhibition by l-aspartate. Acetyl-CoA specifically protects rat liver pyruvate carboxylase against inactivation by trinitrobenzene sulfonate whereas the substrates have little effect on the rate of inactivation. The pseudo-first order rate constant for the inactivation process is a sigmoid function of [acetyl-CoA] (n = 2.4) and 50% reduction in the value of this rate constant is observed in the presence of 310 µm acetyl-CoA. Studies of the specificity of inactivation and the properties of the modified enzyme suggest that modification of 1 lysine residue per catalytic site is responsible for the observed effect in accord with earlier studies on inactivation of mammalian kidney pyruvate carboxylase by this reagent (Keech, D. B., and Farrant, R. K. (1968) Biochim. Biophys. Acta, 151, 493). These data provide the first direct indication that activation of these pyruvate carboxylases by acetyl-CoA results from interaction of this modifier at an allosteric site, which, in the case of the enzyme from Saccharomyces cerevisiae, differs from that occupied by l-aspartate.