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.

Regulation of the structure and activity of pyruvate carboxylase by acetyl CoA

“…From these studies and those by Phillips, et al [41], it was determined that the partial reactions catalysed by chicken and rat liver PC were all activated to some extent by the addition of acetyl CoA, but those reactions where the mechanism requires the formation of the putative carboxyphosphate intermediate and subsequent formation of carboxybiotin are more, if not completely, dependent on the presence of acetyl CoA. Attwood and Graneri [38] examined the effects of acetyl CoA on steady-state kinetics of the HCO 3 − -dependent ATP cleavage catalysed by chicken liver PC.…”

“…The binding of pyruvate signals the movement of carboxybiotin from the first catalytic site in the BC domain (subsite 1) to the CT domain (subsite 2) [52]. The enzyme-carboxybiotin complex was isolated in PC from sheep and chicken (Scrutton et al, 1965; Goodall et al, 1981; Attwood et al, 1984; Attwood and Wallace, 1986; Phillips et al, 1992; Attwood, 1993)[41, 47, 50, 53-55]. The isolated enzyme-carboxybiotin complex efficiently converted pyruvate to oxaloacetate in the absence of MgATP and HCO 3 − , both in the presence and absence of acetyl CoA.…”

Structure, function and regulation of pyruvate carboxylase

“…The observation that PCs from sheep kidney and chicken liver are also capable of catalysing the phosphorylation of ADP from carbamoyl phosphate provides further support for such an intermediate [69,70]. Support for this proposed mechanism was provided by the isolation of a putative enzyme-bound carboxyphosphate intermediate [71]. In the presence of acetyl-CoA, the carboxy group is subsequently transferred to biotin to form carboxybiotin, which is the product of the first partial reaction [71,72].…”

“…Support for this proposed mechanism was provided by the isolation of a putative enzyme-bound carboxyphosphate intermediate [71]. In the presence of acetyl-CoA, the carboxy group is subsequently transferred to biotin to form carboxybiotin, which is the product of the first partial reaction [71,72]. Further evidence favouring the formation of a carboxyphosphate intermediate is to be found in the similarity of the primary structure of biotin carboxylase to that region of carbamoyl-phosphate synthase (CPS ; EC 6.3.5.5)…”

Biochemical characterization of plant biotin-containing enzymes

“…In the first-half reaction, in which ATP is hydrolyzed and biotin is carboxylated, probably via a carboxy-phosphate intermediate (Kaziro et al, 1962;Climent and Rubio, 1986;Tipton and Cleland, 1988;Ogita and Knowles, 1988;Phillips et al, 1992), the first two products, ADP and Pi are released from the enzyme. The "charged", carboxylated enzyme (E' in Scheme H) then reacts with Mc-CoA to form the final product 3-methylglutaconyl-CoA.…”