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Query: EC:4.1.1.32 (phosphoenolpyruvate carboxykinase)
 4,204 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The kinetic mechanism of yeast phosphoenolpyruvate carboxykinase, in the physiological direction, has been determined. Product inhibition using KHCO3 showed competitive inhibition, when both oxalacetate (OAA) and ATP were varied. Phosphoenolpyruvate showed noncompetitive inhibition against OAA, and competitive inhibition with respect to ATP. Conversely, ADP showed competitive inhibition against OAA and noncompetitive inhibition vs. ATP. Dead-end inhibition studies with beta-sulfopyruvate showed competitive inhibition against OAA and noncompetitive inhibition vs. ATP. Ethene-ATP exhibited competitive inhibition against ATP and noncompetitive inhibition with respect to OAA. These results are consistent with a random Bi-Ter mechanism with the formation of two abortive complexes: enzyme-ATP-ADP and enzyme-OAA-PEP.
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PMID:The kinetic mechanism of yeast phosphoenolpyruvate carboxykinase. 842 23

Avian mitochondrial phosphoenolpyruvate carboxykinase (PEPCK) was incubated with Co2+ and H2O2 to form a stable Co3+-PEPCK complex. PEPCK, similarly incubated with H2O2 and either Mg2+ or Mn2+, resulted in no significant loss in activity over 30 min. PEPCK, incubated with Co2+ and H2O2 at pH 7.4, showed rapid inhibition as observed by a 40% decrease in activity after 5 min. The loss of activity is linear with the incorporation of cobalt into PEPCK, resulting in 15-25% activity for the stoichiometric Co3+-PEPCK complex. The incorporation of and inhibition by Co3+ is protected by PEP and GTP (ITP). Treatment of the Co3+-PEPCK complex with beta-mercaptoethanol results in a loss of cobalt and full recovery of activity. The reduction and reactivation are protected by PEP and GTP (ITP). EPR, PRR, circular dichroism, and fluorescence studies all indicate that Co3+ has been selectively incorporated into the cation site of PEPCK, resulting in a catalytically active enzyme-cation species. The substrates form Michaelis complexes with Co3+-PEPCK, and the catalytic reaction occurs as a second sphere complex as previously suggested [Lee & Nowak (1984) Biochemistry 23, 6506); Duffy & Nowak (1985) Biochemistry 24, 1152]. Proteolytic digestion of the Co3+-PEPCK complex and isolation of the cobalt-containing peptide by reverse phase HPLC were performed to identify the location of the cation binding site. From mass, amino acid composition, and sequence analyses of the isolated cobalt-peptide, the region Thr276-Lys301 is responsible for metal chelation. This very homologous region, located in the central portion of PEPCK, contains two highly conserved aspartic acids, Asp295 and Asp296, that are the only feasible metal binding ligands.
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PMID:Formation and characterization of an active phosphoenolpyruvate carboxykinase-cobalt(III) complex. 911 19

Chicken liver phosphoenolpyruvate carboxykinase (PEPCK) was rapidly inactivated by micromolar concentrations of ferrous sulfate in the presence of ascorbate at pH 7.4. Omitting ascorbate or replacing the Fe2+ with Mn2+ or Mg2+ gives no inactivation. Mn2+, Mg2+, or Co2+ at 100-fold molar excess over Fe2+ offered complete protection from Fe2+/ascorbate-induced inactivation. The substrates PEP and GTP, but not OAA, GDP, or CO2, offered full protection from inactivation. The addition of 5 mM EDTA stopped further inactivation of the enzyme. Thermodynamic studies indicate that the inactive enzyme no longer binds Mn2+ but still had high affinity for GTP indicating that the inactivation process was specific for the metal site. A decrease in cysteine content was observed over time following PEPCK treatment with Fe2+ and ascorbate. The apparent first-order rate constant for free sulfhydryl loss (0.085 +/- 0.005 min-1) is similar to the apparent first-order rate constant for inactivation (0.067 +/- 0.005 min-1). Amino acid composition analysis revealed that cysteic acid was generated upon Fe2+/ascorbate addition to PEPCK. Native chicken liver PEPCK has an Mr of 67 kDa. SDS-PAGE of the inactivated enzyme showed the presence of two new bands at 31.7 and 35.3 kDa indicating that PEPCK was specifically cleaved at a single site. The rate of cleavage was slower than the rate of inactivation and fully inactivated enzyme was only 50% cleaved. The Fe2+/ascorbate-catalyzed inactivation was not solely due to protein cleavage. The protein fragments generated by cleavage were separated by C4 reverse phase HPLC. The cleavage exposed a new N-terminus which was identified to be the 35.3 kDa C-terminal half of PEPCK. Sequencing of the fragments indicated that the site of cleavage was between Asp296 and Ile297. These results indicate that Asp296 is involved in metal chelation. This agrees with previous studies [Hlavaty, J. J., & Nowak, T. (1997) Biochemistry 36, 3389-3403] that suggested that Asp295 and Asp296 are involved in metal binding.
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PMID:Affinity cleavage at the metal-binding site of phosphoenolpyruvate carboxykinase. 939 80

Lys606, one of the two highly conserved lysine residues in maize C4-form phosphoenolpyruvate carboxylase (PEPC), was converted to Asn, Glu or Arg by site-directed mutagenesis. Resulted mutant enzymes expressed using pET system [Dong, L.-Y. et al. (1997) Biosci. Biotech, Biochem. 61:545] were purified by one step procedure through nickel-chelate affinity chromatography to a purity of about 95%. The replacement of Lys606 by Arg had little effect on the kinetic and allosteric properties of the resulting mutant enzyme. In contrast, the maximum velocities (Vmax) were decreased to 22% and 2% of that of wild-type PEPC upon the substitution of Lys606 by Asn and Glu, respectively. The value of S0.5(HCO3-) was increased 21-25 fold by the replacements, whereas the S0.5(Mg2+) and S0.5(PEP) values were increased only 5-8 fold. The extents of activation of mutant enzymes by glucose 6-phosphate and glycine were 2 to 3-fold higher than those of wild-type enzyme. The mutant enzymes showed less sensitivity to malate inhibition, compared with the wild-type enzyme. The results suggested that the Lys606 is not obligatory for the enzyme activity, but may be involved in the bicarbonate-binding and contribute somehow to the allosteric regulatory properties.
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PMID:Effects of site-directed mutagenesis of conserved Lys606 residue on catalytic and regulatory functions of maize C4-form phosphoenolpyruvate carboxylase. 952 66

A full-length cDNA for maize root-form phosphoenolpyruvate carboxylase (PEPC) was isolated. In the coding region, the root-form PEPC showed 76 and 77% identity with the C4- and C3-form PEPCs of maize, respectively, at the nucleotide level. At the amino acid level, the root-form was 81 and 85% identical to the C4- and C3-form PEPCs, respectively. The entire coding region was inserted into a pET32a expression vector so that it was expressed under the control of T7 promoter. The purified recombinant root-form PEPC had a Vmax value of about 28 mumol min-1 (mg protein)-1 at pH 8.0. The K(m) values of root-form PEPC for PEP and Mg2+ were one-tenth or less of those of C4-form PEPC when assayed at either pH 7.3 or 8.0, while the value for HCO3- was about one-half of that of C4-form PEPC at pH 8.0. Glucose 6-phosphate and glycine had little effect on the root-form PEPC at pH 7.3; they caused two-fold activation of the C4-form PEPC. The Ki (L-malate) values at pH 7.3 were 0.12 and 0.43 mM for the root- and C4-form PEPCs, respectively. Comparison of hydropathy profiles among the maize PEPC isoforms suggested that several stretches of amino acid sequences may contribute in some way to their characteristic kinetic properties. The root-form PEPC was phosphorylated by both mammalian cAMP-dependent protein kinase and maize leaf protein kinase, and the phosphorylated enzyme was less sensitive to L-malate.
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PMID:Cloning, expression, and characterization of a root-form phosphoenolpyruvate carboxylase from Zea mays: comparison with the C4-form enzyme. 978 61

Acute intravenous infusions of leptin markedly alter hepatic glucose fluxes (Rossetti, L., Massillon, D., Barzilai, N., Vuguin, P., Chen, W., Hawkins, M., Wu, J., and Wang, J. (1997) J. Biol. Chem. 272, 27758-22763). Here we examine whether intracerebroventricular (ICV) leptin administration regulates peripheral and hepatic insulin action. Recombinant mouse leptin (n = 14; 0.02 or 1 microgram/kg.h) or vehicle (n = 9) were administered ICV for 6 h to conscious rats, and insulin action was determined by insulin (3 milliunits/kg.min) clamp and tracer dilution techniques. During physiologic hyperinsulinemia (approximately 65 microunits/ml), the rates of glucose uptake (Rd, 20.1 +/- 0.6 and 23.1 +/- 0.7 versus 21.7 +/- 0.6 mg/kg.min; p = NS), glycolysis and glycogen synthesis were similar in rats receiving low- and high-dose leptin versus vehicle. ICV leptin resulted in a 2-3-fold increase in hepatic phosphoenolpyruvate carboxykinase mRNA levels. Glycogenolysis and PEP-gluconeogenesis (2.1 +/- 0.3 mg/kg. min) contributed similarly to endogenous glucose production (GP) in the vehicle-infused group. However, gluconeogenesis accounted for approximately 80% of GP in both groups receiving ICV leptin, while hepatic glycogenolysis was markedly suppressed (0.7 +/- 0.3 and 1.2 +/- 0.3 versus 2.2 +/- 0.4 mg/kg.min, in rats receiving low- and high-dose leptin versus vehicle, respectively; p < 0.01). In summary, short-term ICV leptin administration: 1) failed to affect peripheral insulin action, but 2) induced a striking re-distribution of intrahepatic glucose fluxes. The latter effect largely reproduced that of leptin given systemically at much higher doses. Thus, the regulation of hepatic glucose fluxes by leptin is largely mediated via its central receptors.
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PMID:Intracerebroventricular leptin regulates hepatic but not peripheral glucose fluxes. 981 20

This study provides the first comparative analysis of phosphoenolpyruvate carboxylase isoforms (PEPc; EC 4.1.1.31) in an obligate crassulacean acid metabolism (CAM) plant, Vanilla planifolia Salisb. (Orchidaceae). Nocturnal CO2 fixation and malate accumulation by the leaves and the green stem show that these organs perform CAM. The chloroplast-containing aerial roots, however, exhibit C3 photosynthesis. The catalytic activity of PEPc was highest in the leaves compared with the stem and aerial roots. The Km (PEP) and Ki (malate) were similar in the PEPc extracted from leaf and aerial roots, and significant higher in stem. cDNA was obtained from those tissues and also from the soil-grown roots, and various cDNA clones were detected and amplified by means of RT-PCR and RACE-PCR. The amino-acid sequences of the PEPc isoforms deduced from the cDNA showed a great degree of homology, and Southern blot analysis suggests that the encoding genes form a small multigene family of at least two members. One PEPc isoform (PpcV1) is assumed to be related to CAM because, as shown by northern blot analysis, it is mainly expressed in the CAM-performing organs, i.e. in the leaves and the stem. A further isoform (PpcV2) was identified in the soil-grown roots and aerial roots, but northern blots show that to some extent PpcV2 is also expressed in the leaf and the stem tissues. Thus, it is assumed that PpcV2 encodes the housekeeping isoform of PEPc. Altogether, the present study provides support in favour of the view that isoforms of PEPc are related to specific functions.
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PMID:Identification of phosphoenolpyruvate carboxylase isoforms in leaf, stem and roots of the obligate CAM plant Vanilla planifolia Salib. (Orchidaceae): a physiological and molecular approach. 986 26

Chicken liver phosphoenolpyruvate carboxykinase (PEPCK) requires two divalent cations for activity. One cation activates the enzyme through a direct interaction with the protein at site n(1). The second cation, at site n(2), acts in the cation-nucleotide complex that serves as a substrate. The Co(3+)(n(1))-PEPCK and Cr(3+)(n(1))-PEPCK complexes were used to examine the kinetic, mechanistic, and binding properties of the n(2) metal. EPR studies performed on the Co(3+)(n(1))-PEPCK-GTP complex yielded a stoichiometry of 1 mol of Mn(2+) bound per mole of Co(3+)(n(1))-PEPCK-GTP with a K(D) of 5 microM. PRR studies show a significant enhancement for the Co(3+)(n(1))-PEPCK-Mn(2+)(n(2))-GDP complex. A change in enhancement in the presence of PEP suggests that PEP interacts with the second metal ion. The distance between Mn(2+) at site n(2) on PEPCK and the cis and trans protons and the (31)P of PEP are 7.0, 7.5, and 4.8 A, respectively, as measured by high-resolution NMR. PRR studies of the Co(3+)(n(1))-PEPCK-Mn(2+)(n(2))-GTP and Co(3+)(n(1))-PEPCK-Mn(2+)(n(2))-GDP complexes as a function of frequency (omega(I)) were used to estimate the hydration number of the n(2) metal to be between 0.5 and 0.7. The metal-metal distance for the M(n(1))-PEPCK-M(n(2))-GTP complex is approximately 8.3 A, and the distance for the M(n(1))-PEPCK-M(n(2))-GDP complex is 9.2 A. The change in the metal-metal distance suggests a conformational change at the active site of PEPCK occurs during catalysis. The Co(3+)(n(1))-PEPCK complex was incubated with Co(2+), GTP, and H(2)O(2) to create a doubly labeled and inactive Co(3+)(n(1))-PEPCK-Co(3+)(n(2))-GTP complex. The Co(3+)(n(1))-PEPCK-Co(3+)(n(2))-GTP complex was digested by LysC, and two cobalt-containing peptides were purified using RP-HPLC. Amino acid sequencing of the second cobalt-containing peptide points to the region of Tyr57-Lys76 of PEPCK. Asp66, Asp69, and Glu74 are all feasible ligands to the site n(2) metal.
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PMID:Characterization of the second metal site on avian phosphoenolpyruvate carboxykinase. 1068 18

Quantifying the contribution of the various skeletal muscle fiber types toward lactate disposal has proven elusive. In part, this can be attributed to the lack of adequate preparations for the study of all potential metabolic pathways involved. Toward this end our laboratory developed several perfused muscle preparations that are homogeneous for specific fiber types. This paper briefly reviews our findings regarding the influence of fiber type on lactate disposal in resting skeletal muscle and the metabolic pathways involved. Perfusing over a range of lactate concentrations, 1-12 mM, all fiber types were shown to switch from net production at low lactate concentrations to net consumption at higher concentrations. This transition occurred at lower lactate concentrations for Type I and IIa fibers, when compared with IIb fibers. For Type I and IIa fibers oxidation was observed to be the primary route of disposal accounting for approximately 50% of the lactate removed. For all fiber types, transamination was a significant pathway for the disposal of lactate carbon, whereas glyconeogenesis was the primary pathway for disposal in Type IIb fibers. The glyconeogenic capacity was quantitatively similar for Type IIa and IIb fibers but was negligible for Type I fibers. The pathway for glyconeogenesis in skeletal muscle was shown to be substantially different from that employed in hepatic glyconeogenesis. Results indicated that neither the TCA cycle nor phosphoenolpyruvate carboxykinase is involved in skeletal muscle glyconeogenesis. Our findings suggested that PEP formation in skeletal muscle glyconeogenesis occurs by "reversal" of the pyruvate kinase reaction.
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PMID:Quantitative assessment of pathways for lactate disposal in skeletal muscle fiber types. 1077 96

C4 phosphoenolpyruvate carboxylases have evolved from ancestral C3 isoforms during the evolution of angiosperms and gained distinct kinetic and regulatory properties compared with the C3 isozymes. To identify amino acid residues and/or domains responsible for these C4-specific properties the C4 phosphoenolpyruvate carboxylase of Flaveria trinervia (C4) was compared with its orthologue in the closely related C3 plant Flaveria pringlei. Reciprocal enzyme chimera were constructed and the kinetic constants, K(0.5) and k(cat), as well as the Hill coefficient, h, were determined for the substrate phosphoenolpyruvate both in the presence and absence of the activator glucose 6-phosphate. By this approach two regions were identified which determined most of the kinetic differences of the C4 and C3 ppcA phosphoenolpyruvate carboxylases with respect to the substrate PEP. In addition, the experiments suggest that the two regions do not act additively but interact with each other. The region between amino acids 296 and 437 is essential for activation by glucose 6-phosphate. The carboxyl-terminal segment between amino acids 645 and 966 contains a C4 conserved serine or a C3 invariant alanine at position 774 in the respective enzyme isoform. Site-directed mutagenesis shows that this position is a key determinant for the kinetic properties of the two isozymes.
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PMID:Evolution of C4 phosphoenolpyruvate carboxylase in Flaveria, a conserved serine residue in the carboxyl-terminal part of the enzyme is a major determinant for C4-specific characteristics. 1087 30


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