Mammalian and avian liver phosphoenolpyruvate carboxykinase. Alternate substrates and inhibition by analogues of oxaloacetate.

Ash, David E.; Emig, Frances A.; Chowdhury, Soheli A.; Satoh, Yoshitake; Schramm, Vern L.

doi:10.1016/s0021-9258(19)39124-0

Cited by 47 publications

(33 citation statements)

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“…In addition to this physiologically important reaction, the decarboxylation of OAA to pyruvate + CO 2 and a pyruvate kinase-like activity have been reported in Saccharomyces cereVisiae and other PEP carboxykinases (2)(3)(4). In all cases examined, the reaction catalyzed by PEP carboxykinases follows a sequential kinetic mechanism (5,6) and a direct transfer of the γ-phosphate of the nucleoside triphosphate to OAA has been proposed for the guinea pig liver mitochondrial and rat liver cytosolic enzymes (8,9).…”

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confidence: 92%

The Strongly Conserved Lysine 256 of Saccharomyces cerevisiae Phosphoenolpyruvate Carboxykinase Is Essential for Phosphoryl Transfer

et al. 1998

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Lysine 256, a conserved amino acid of Saccharomycescerevisiae phosphoenolpyruvate (PEP) carboxykinase located in the consensus kinase 1a sequence of the enzyme, was changed to alanine, arginine, or glutamine by site-directed mutagenesis. These substitutions did not result in gross changes in the protein structure, as indicated by circular dichroism, tryptophan fluorescence spectroscopy, and gel-exclusion chromatography. The three variant enzymes showed almost unaltered Km for MnADP but about a 20 000-fold decrease in Vmax for the PEP carboxylation reaction, as compared to wild-type PEP carboxykinase. The variant enzymes presented oxaloacetate decarboxylase activity at levels similar to those of the native protein; however, they lacked pyruvate kinase-like activity. The dissociation constant for the enzyme-MnATP complex was 1.3 +/- 0.3 microM for wild-type S. cerevisiae PEP carboxykinase, and the corresponding values for the Lys256Arg, Lys256Gln, and Lys256Ala mutants were 2.0 +/- 0.6 microM, 17 +/- 2 microM, and 20 +/- 6 microM, respectively. These results collectively show that a positively charged residue is required for proper binding of MnATP and that Lys256 plays an essential role in transition state stabilization during phosphoryl transfer for S. cerevisiae PEP carboxykinase.

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confidence: 92%

The Strongly Conserved Lysine 256 of Saccharomyces cerevisiae Phosphoenolpyruvate Carboxykinase Is Essential for Phosphoryl Transfer

et al. 1998

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“…The reversible inhibition of PEPCK by various small molecules has been examined by many groups over the past 50 years. These studies have largely focused on either analogues of PEP/OAA, − tryptophan metabolites, − or nucleotide analogues. , Focusing upon the PEP/OAA binding site, in 2008 Stiffin et al used various commercially available small molecules to understand the physicochemical properties required for inhibition of PEPCK. , These small molecules were selected with the rationale that they were analogues of OAA and PEP. Structural and kinetic studies demonstrated that two inhibitors, sulfoacetate and oxalate, defined two partially overlapping binding pockets in the PEP/OAA binding site; these two sites were defined as the inner and outer subsites.…”

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confidence: 99%

Characterization of 3-[(Carboxymethyl)thio]picolinic Acid: A Novel Inhibitor of Phosphoenolpyruvate Carboxykinase

et al. 2019

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Phosphoenolpyruvate carboxykinase (PEPCK) has traditionally been characterized for its role in the first committed step of gluconeogenesis. The current understanding of PEPCK’s metabolic role has recently expanded to include it serving as a general mediator of tricarboxylic acid cycle flux. Selective inhibition of PEPCK in vivo and in vitro has been achieved with 3-mercaptopicolinic acid (MPA) (K i ∼ 8 μM), whose mechanism of inhibition has been elucidated only recently. On the basis of crystallographic and mechanistic data of various inhibitors of PEPCK, MPA was used as the initial chemical scaffold to create a potentially more selective inhibitor, 3-[(carboxymethyl)thio]picolinic acid (CMP), which has been characterized both structurally and kinetically here. These data demonstrate that CMP acts as a competitive inhibitor at the OAA/PEP binding site, with its picolinic acid moiety coordinating directly with the M1 metal in the active site (K i ∼ 29–55 μM). The extended carboxy tail occupies a secondary binding cleft that was previously shown could be occupied by sulfoacetate (K i ∼ 82 μM) and for the first time demonstrates the simultaneous occupation of both OAA/PEP subsites by a single molecular structure. By occupying both the OAA/PEP binding subsites simultaneously, CMP and similar molecules can potentially be used as a starting point for the creation of additional selective inhibitors of PEPCK.

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“…A requirement for phosphoryl transfer could be occupation of the binding site for the carboxylic acid. Addition of oxalate, a competitive inhibitor with respect to oxaloacetate with a value of <20 µ (Ash et al, 1990), is unable to support positional isotope exchange. The sum of these results indicates that phosphoryl transfer to enzyme does not occur in the normal catalytic cycle of mitochondrial or cytosolic P-enolpyruvate carboxykinases.…”

Section: Discussionmentioning

confidence: 99%

“…for the enzyme with MgGTP and 20 µ free MnCl2 (Schramm et al, 1981). The kinetic constant of 2 µ for the chicken liver enzyme is based on experiments with MgGTP fixed at 50 and 100 µ in the presence of 50 µ MnCl2 (Ash et al, 1990). The dashed arrows for the chicken enzyme indicate that these steps are kinetically insignificant.…”

Section: Discussionmentioning

confidence: 99%

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Isotope trapping and positional isotope exchange with rat and chicken liver phosphoenolpyruvate carboxykinases

Chen¹,

Sato²,

Schramm³

1991

Biochemistry

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Isotope-trapping studies of the enzyme.MgGTP complex were carried out with rat liver cytosolic and chicken liver mitochondrial phosphoenolpyruvate carboxykinases. For the rat liver enzyme, MgGTP was partially trapped from both E.MgGTP and E.MgGTP.OAA complexes, consistent with a steady-state random mechanism. For the chicken liver enzyme, MgGTP was 100% trapped from the E.MgGTP.OAA complex, consistent with a steady-state ordered mechanism. The rate constants for the interaction of MgGTP with the free enzymes are approximately 10(7) M-1 S-1, somewhat lower than the diffusion limit for association. The dissociation rate for the enzyme.MgGTP complexes is 26-92 s-1, reflecting a tightly bound complex with high commitment to catalysis in the presence of oxaloacetate. Positional isotope-exchange studies were also carried out with phosphoenolpyruvate carboxykinases from rat and chicken. No exchange if the beta gamma-18O in [beta gamma-18O, gamma-18O3]GTP to form [beta-18O, gamma-18O3]GTP was detected in the absence of oxaloacetate. In the presence of oxaloacetate, no positional isotope exchange of [beta gamma-18O, gamma-18O3]GTP was detected during initial rate conditions. The results indicate that at least one of the products dissociates rapidly from the E.MgGDP.PEP.CO2 complex relative to the net rate of MgGTP formation from the E.MgGDP.PEP.CO2 complex. A rapid equilibrium between the central complexes in which the beta-phosphoryl of GDP is restricted with respect to torsional rotation cannot be excluded but is unlikely on the basis of the relative rates of catalysis and torsional rotation. The addition of Mn2+, an activator of phosphoenolpyruvate carboxykinase, did not influence the positional isotope-exchange results.(ABSTRACT TRUNCATED AT 250 WORDS)

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Mammalian and avian liver phosphoenolpyruvate carboxykinase. Alternate substrates and inhibition by analogues of oxaloacetate.

Cited by 47 publications

References 46 publications

The Strongly Conserved Lysine 256 of Saccharomyces cerevisiae Phosphoenolpyruvate Carboxykinase Is Essential for Phosphoryl Transfer

The Strongly Conserved Lysine 256 of Saccharomyces cerevisiae Phosphoenolpyruvate Carboxykinase Is Essential for Phosphoryl Transfer

Characterization of 3-[(Carboxymethyl)thio]picolinic Acid: A Novel Inhibitor of Phosphoenolpyruvate Carboxykinase

Isotope trapping and positional isotope exchange with rat and chicken liver phosphoenolpyruvate carboxykinases

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