Biochemistry zyxwvu 1992,31, zyxwvu 9445-9450 9445 zyxwvutsrqp Isolation of a Carboxyphosphate Intermediate and the Locus of Acetyl-coA Action in the Pyruvate Carboxylase Reaction? Nelson F. B. Phillips,: Mark A. Snoswell, Anne Chapman-Smith, D. Bruce Keech, and John C. Wallace' Department of Biochemistry, University of Adelaide, Adelaide, South Australia, Australia Received March 31, 1992; Revised Manuscript Received June 26, 1992 zyxwvutsrqponmlkjihgfedcbaZYXWVU ABSTRACT: When chicken liver pyruvate carboxylase was incubated with either H14C03- or y- [32P]ATP, a labeled carboxyphospho-enzyme intermediate could be isolated. Thecomplex was catalytically competent, as determined by its subsequent ability to transfer either 14C02 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 zyxwvut [ 14C]carboxyphospho+nzyme and the~arboxy[~~P]phospho- enzyme had similar half-lives. Acetyl-coA was shown to be involved in the conversionof 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. zyxwvutsrqponml As part of an ongoing investigationinto the reaction pathway of the biotin-dependent enzyme, pyruvate carboxylase (PC;' EC 6.4.1. l), and the locus of action of the allosteric activator, acetyl-coA, we have considered the following established information: (a) Under equilibriumconditions, PC catalyzes four clearly defined isotope exchange reactions (Scrutton et al., 1965; McClure et al., 1971a; Ashman & Keech, 1975) Mg2+ ATP + ADP (1) ATP + Pi (2) Mg2+,acetyl-CoA pyruvate + oxalacetate (3) HC0,- * oxalacetate (4) acetyl-coA ADP, Pi While reactions 1 and 3 can proceed in the absence of acetyl- CoA, the presence of the activator increases the rate of all four isotope exchange reactions (Ashman et al., 1972; At- twood & Keech, 1985). (b) When assayed under identical conditions, reactions 1 and 2 proceed at the same rate (McClure et al., 1971b), indicating that, although the observed ATP/ADP exchange is not inhibited by avidin (Ashman & Keech, 1975), it is a part of the main reaction sequence. However, the two processes can be uncoupled by a partial denaturation of the enzyme (Wallace et al., 1985). ?This work was supported by a grant (D278/15094) from the Australian Research Grants Commission (to D.B.K. and J.C.W.). N.F.B.P. was the recipient of a University of Adelaide Postgraduate Research Award. * Correspondence: Department of Biochemistry, University of Ade- laide, GPO Box 498, Adelaide SA 5001, Australia. * Present address: Departments of Biochemistry and Medicine, Case Western Reserve University, Cleveland, OH 44106. I Abbreviations: PC, pyruvate carboxylase (EC 6.4.1.1);PEI, poly- ethylenimine. On the basis of these data, the overall reaction catalyzed by PC ATP + HC0,- + pyruvate can be broken down into the following minimal steps: Mg2+. acetyl-coA + ADP + Pi + oxalacetate (5) zyxwvutsrq Mg2+ ENZ-biotin + ATP = ATP-ENZ+iotin ( 6) ENZ-biotin-CO,- + pyruvate = ENZ-biotin + oxalacetate (9) In support of this sequence of events for the carboxylationof the biotin moiety there is the ample evidence that biotin is not directly involved in the cleavage of ATP [see Knowles (1989)l. Climent and Rubio (1986) have shown that biotin does not participate in HCO3--dependent ATP cleavage for biotin car- boxylase from Escherichia coli. Additionally, the experiments of Kaziro et al. (1962), using HC'*03- to investigate the mechanism of propionyl-CoA carboxylase (EC 6.4.1,3), showed that bicarbonate participates directly in the cleavage of the &y phosphoryl bond of ATP (eq 7). In this paper, we demonstrate the formation of a carboxy- phosphateenzyme complex of PC prior to the carboxylation of biotin (see eq 8) and show that one of the effects of acetyl- CoA occurs during the subsequent transfer of the 'activated" carboxyl group to the biotin moiety. A preliminary account of this work has been presented elsewhere (Wallace et al., 1985). MATERIALS AND METHODS Preparation and Assay of Pyruvate Carboxylase. Chicken liver pyruvate carboxylasewas prepared as described by Goss 0006-2960/92/043 1 -9445$03.00/0 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 0 1992 American Chemical Society