Isolation of a Carboxyphosphate Intermediate and the Locus of Acetyl-CoA Action in the Pyruvate Carboxylase Reaction

Abstract

When chicken liver pyruvate carboxylase was incubated with either H14C03 or ᵧ-[32P] ATP, a labeled carboxyphosphoenzyme intermediate could be isolated. The complex was catalytically competent, as determined by its subsequent ability to transfer either 14CO2 to pyruvate or 32P to ADP. While the carboxyphosphoenzyme complex was inherently unstable and the stoichiometry of the transfer was variable depending on experimental conditions, both the [ 14C] carboxyphosphoenzyme and the carboxy [32P] phosphoenzyme had similar half-lives. Acetyl-CoA was shown to be involved in the conversion of the carboxyphosphoenzyme complex to the more stable carboxybiotin-enzyme species, which was consistent with the effects of acetyl-CoA on isotope exchange reactions involving ATP. We were unable to detect the formation of a phosphorylated biotin derivative during the ATP cleavage reaction. In the presence of K+ and at pH 9.5, the acetyl-CoA-independent activity of chicken liver pyruvate carboxylase approached 2% of the acetyl-CoA-stimulated rate, which represents a 30-fold increase on previously reported activity for this enzyme. © 1992, American Chemical Society. All rights reserved.