title: 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 H14CO3- or gamma-[32P]ATP, a labeled carboxyphospho-enzyme 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 carboxyphospho-enzyme complex was inherently unstable and the stoichiometry of the transfer was variable depending on experimental conditions, both the [14C]carboxyphospho-enzyme and the carboxy[32P]phospho-enzyme had similar half-lives. Acetyl-CoA was shown to be involved in the conversion of the carboxyphospho-enzyme 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.
authors.affiliation: Department of Biochemistry, University of Adelaide, Australia.
authors.forename: N F
authors.initials: NF
authors.lastname: Phillips
authors.forename: M A
authors.initials: MA
authors.lastname: Snoswell
authors.forename: A
authors.initials: A
authors.lastname: Chapman-Smith
authors.forename: D B
authors.initials: DB
authors.lastname: Keech
authors.forename: J C
authors.initials: JC
authors.lastname: Wallace
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001710:Biotin / Q000378:metabolism; D002245:Carbon Dioxide / Q000378:metabolism; D002264:Carboxylic Acids / Q000378:metabolism; D002384:Catalysis; D002645:Chickens; D008099:Liver / Q000201:enzymology; D046911:Macromolecular Substances; D010710:Phosphates / Q000378:metabolism; D011766:Pyruvate Carboxylase / Q000378:metabolism*
chemical_list: D002264:Carboxylic Acids; D046911:Macromolecular Substances; D010710:Phosphates; D002245:Carbon Dioxide; D001710:Biotin; D000105:Acetyl Coenzyme A; D000255:Adenosine Triphosphate; D011766:Pyruvate Carboxylase