Liver acetyl CoA carboxylase: insight into the mechanism of activation by tricarboxylic acids and acetyl CoA.

Abstract

Isocitrate (or citrate) is a positive allosteric effector of the Vm type according to the classification of Monod et al. The mechanism by which isocitrate promotes catalytic activity was explored by studying malonyl CoA and enzyme-CO2- decarboxylation since these constitute minimal reactions which are uncomplicated by a requirement for ATP, ADP, Pi, HCO3-, or Mg2+. While these decarboxylations are comparatively slow with respect to the over-all acetyl CoA carboxylation reaction, variations in their rates are indicative of the reactivity of the labile-carboxy group of enzyme-CO2-. It was found that isocitrate activates the decarboxylation of malonyl CoA and also shortens the half life of enzyme-CO2-. In addition to isocitrate, the decarboxylation of malonyl CoA exhibits a nearly absolute requirement for acetyl CoA. Furthermore, acetyl CoA alone markedly activates the decarboxylation of enzyme-CO2- ([forward arrow] enzyme + CO2) and in combination with isocitrate leads to at least a 24-fold increase in the rate of this decarboxylation. This strongly suggests that both isocitrate and acetyl CoA increase the reactivity of the labile carboxy group of the carboxylated enzyme. These results are interpretable in terms of a mechanism in which it is visualized that the conformational change induced by isocitrate brings a proton-donating group into closer proximity with the ureido carbonyl of the biotinyl prosthetic group. Protonation of the ureido carbonyl oxygen would lead to polarization of the N-carboxy C-N bond. This promotes labilization of the N-carboxy group and accounts for the activation of both decarboxylation and transfer to acetyl CoA. Since acetyl CoA causes a limited, but reproducible, aggregation of the carboxylase it may, like isocitrate, lead to conformational changes which have the same net effect on the relationship of the biotinyl moiety to adjacent groups. That activation by isocitrate and acetyl CoA is accompanied by conforma-tional changes in the vicinity of the biotinyl prosthetic group is supported by the protection from avidin inactivation afforded by these activators. This suggests that in the presence of isocitrate (or citrate) and acetyl CoA the biotinyl prosthetic group is less accessible to avidin possibly because of greater shielding by neighboring groups.