Pyruvate Carboxylase

Pyruvate carboxylase purified from chicken liver shows an essentially absolute requirement for activation by an acyl coenzyme A in all preparations and under all conditions tested thus far. The enzyme is activated by formyl-, acetyl-, propionyl-, crotonyl-, and iso-butyryl-CoA, but carboxylated analogues of the activating acyl derivatives of CoA and certain other acetyl thioesters are potent competitive inhibitors with respect to acetyl-CoA. Examination of the initial reaction rate as a function of activator concentration reveals a sigmoid relationship, suggesting that more than 1 molecule of activator per active site is required for the activation process or that cooperative interactions between the bound activator molecules occur. Inactivation analyses show an interaction of acetyl-CoA with pyruvate carboxylase, with resultant marked alterations in the tertiary and quaternary structures of the protein as compared with the free enzyme. For example, the rate of inactivation of this biotin enzyme by avidin is markedly increased in the presence of acetyl-CoA concentrations similar to those required for activation of the over-all reaction. Acetyl-CoA also affords almost complete protection of this enzyme against denaturation at 2° or by low concentrations of urea and sodium dodecyl sulfate. These findings are consistent with the proposal that acetyl-CoA and the other activator acyl-CoA derivatives act as allosteric effectors for pyruvate carboxylase. However, acetyl-CoA in the presence or absence of other reaction components has no significant effect either on the sedimentation properties of pyruvate carboxylase in the range from 0.5 to 5.0 mg of protein per ml or on the absorption spectrum of this enzyme. Pyruvate carboxylase catalyzes a very slow hydrolysis of acyl thioesters. An apparent relationship between thioester hydrolysis and the activation of the over-all reaction is suggested by studies of the specificity of hydrolysis and of the effects of other reaction components on the rate of hydrolysis. The evidence appears to exclude the possibility that the activated species of pyruvate carboxylase is an acyl-enzyme, and the observed thioester hydrolysis may therefore be related indirectly to the activation process.