Pyruvate carboxylase: Mechanism of the second partial reaction

Easterbrook‐Smith, Simon B.; Hudson, Peter J.; Goss, Neil H.; Keech, D.B.; Wallace, John C.

doi:10.1016/0003-9861(76)90215-0

Cited by 31 publications

(48 citation statements)

References 27 publications

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“…It was proposed that the binding of pyruvate induces the carboxybiotin to move into the second subsite, where it is destabilized [95]. Goodall et al [96] confirmed this proposal, and also showed that a number of pyruvate analogues can induce the translocation of carboxybiotin to the second subsite.…”

Section: Reaction Mechanismsupporting

confidence: 59%

Structure, function and regulation of pyruvate carboxylase

Jitrapakdee

Wallace

1999

Biochemical Journal

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183

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Pyruvate carboxylase (PC; EC 6.4.1.1), a member of the biotin-dependent enzyme family, catalyses the ATP-dependent carboxylation of pyruvate to oxaloacetate. PC has been found in a wide variety of prokaryotes and eukaryotes. In mammals, PC plays a crucial role in gluconeogenesis and lipogenesis, in the biosynthesis of neurotransmitter substances, and in glucose-induced insulin secretion by pancreatic islets. The reaction catalysed by PC and the physical properties of the enzyme have been studied extensively. Although no high-resolution three-dimensional structure has yet been determined by X-ray crystallography, structural studies of PC have been conducted by electron microscopy, by limited proteolysis, and by cloning and sequencing of genes and cDNA encoding the enzyme. Most well characterized forms of active PC consist of four identical subunits arranged in a tetrahedron-like structure. Each subunit contains three functional domains: the biotin carboxylation domain, the transcarboxylation domain and the biotin carboxyl carrier domain. Different physiological conditions, including diabetes, hyperthyroidism, genetic obesity and postnatal development, increase the level of PC expression through transcriptional and translational mechanisms, whereas insulin inhibits PC expression. Glucocorticoids, glucagon and catecholamines cause an increase in PC activity or in the rate of pyruvate carboxylation in the short term. Molecular defects of PC in humans have recently been associated with four point mutations within the structural region of the PC gene, namely Val145 → Ala, Arg451 → Cys, Ala610 → Thr and Met743 → Thr.

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Section: Reaction Mechanismsupporting

confidence: 59%

Structure, function and regulation of pyruvate carboxylase

Jitrapakdee

Wallace

1999

Biochemical Journal

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183

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“…The ability of pyruvate to easily move in and out of the CT domain active site is also reflected in the increased K m for pyruvate determined for the full forward reaction compared to the K a for pyruvate determined for the stimulation of P i release. A definitive metabolic rationale for the observed lack of stoichiometry between the Re PC-catalyzed MgATP cleavage in the BC domain and pyruvate carboxylation in the CT domain has not been established, although it is suggestive of a regulatory mechanism where elevated levels of pyruvate cause a hyper-compensatory increase in PC efficiency (37). While energetically inefficient at low concentrations of pyruvate, the amount of coupling between cleavage of MgATP and carboxyl transfer still would allow for the production of adequate amounts of oxaloacetate required to maintain essential anaplerotic and gluconeogenic functions (11, 33, 37).…”

Section: Discussionmentioning

confidence: 99%

Activation and Inhibition of Pyruvate Carboxylase from Rhizobium etli

et al. 2011

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While crystallographic structures of the R. etli pyruvate carboxylase (PC) holoenzyme revealed the location and probable positioning of the essential activator, Mg2+, and non-essential activator, acetyl-CoA, an understanding of how they affect catalysis remains unclear. The current steady-state kinetic investigation indicates that both acetyl-CoA and Mg2+ assist in coupling the MgATP-dependent carboxylation of biotin in the biotin carboxylase (BC) domain with pyruvate carboxylation in the carboxyl transferase (CT) domain. Initial velocity plots of free Mg2+ vs. pyruvate were nonlinear at low concentrations of Mg2+ and a nearly complete loss of coupling between the BC and CT domain reactions was observed in the absence of acetyl-CoA. Increasing concentrations of free Mg2+ also resulted in a decrease in the Ka for acetyl-CoA. Acetyl phosphate was determined to be a suitable phosphoryl donor for the catalytic phosphorylation of MgADP, while phosphonoacetate inhibited both the phosphorylation of MgADP by carbamoyl phosphate (Ki = 0.026 mM) and pyruvate carboxylation (Ki = 2.5 mM). In conjunction with crystal structures of T882A R. etli PC mutant cocrystallized with phosphonoacetate and MgADP, computational docking studies suggest that phosphonoacetate could coordinate to one of two Mg2+ metal centers in the BC domain active site. Based on the pH profiles, inhibition studies and initial velocity patterns, possible mechanisms for the activation, regulation and coordination of catalysis between the two spatially distinct active sites in pyruvate carboxylase from R. etli by acetyl-CoA and Mg2+ are described.

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“…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].…”

Section: Locus Of Action Of Acetyl Coa: Pre-steady-state Kineticsmentioning

confidence: 99%

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

Adina-Zada

Zeczycki

Attwood³

2012

Archives of Biochemistry and Biophysics

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In this review we examine the effects of the allosteric activator, acetyl CoA on both the structure and catalytic activities of pyruvate carboxylase. We describe how the binding of acetyl CoA produces gross changes to the quaternary and tertiary structures of the enzyme that are visible in the electron microscope. These changes serve to stabilize the tetrameric structure of the enzyme. The main locus of activation of the enzyme by acetyl CoA is the biotin carboxylation domain of the enzyme where ATP-cleavage and carboxylation of the biotin prosthetic group occur. As well as enhancing reaction rates, acetyl CoA also enhances the binding of some substrates, especially HCO3−, and there is also a complex interaction with the binding of the cofactor Mg2+. The activation of pyruvate carboxylase by acetyl CoA is generally a cooperative processes, although there is a large degree of variability in the degree of cooperativity exhibited by the enzyme from different organisms. The X-ray crystallographic holoenzyme structures of pyruvate carboxylases from Rhizobium etli and Staphylococcus aureus have shown the allosteric acetyl CoA binding domain to be located at the interfaces of the biotin carboxylation and carboxyl transfer and the carboxyl transfer and biotin carboxyl carrier protein domains.

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Pyruvate carboxylase: Mechanism of the second partial reaction

Cited by 31 publications

References 27 publications

Structure, function and regulation of pyruvate carboxylase

Structure, function and regulation of pyruvate carboxylase

Activation and Inhibition of Pyruvate Carboxylase from Rhizobium etli

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

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