Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:4.1.1.49 (phosphoenolpyruvate carboxykinase)
4,654 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ammonium ions stimulated in vitro the activity of PEP carboxylase (PEPC) extracted from dark-adapted leaves of Amaranthus hypochondriacus. Maximum stimulation of 80 to 85% occurred at 50 microM ammonium chloride. There was a marginal inhibition of PEPC at 5 mM ammonium chloride. Among several ions tested, potassium ions stimulated PEPC to a limited extent of about 30%. In presence of ammonium, there was no change either in the sensitivity of enzyme to malate or in the affinity for substrate, PEP. On the other hand, glucose-6-phosphate, an allosteric activator, which stimulated the enzyme by two-fold, could enhance PEPC activity by < 20% in the presence of ammonium. The light-activated form of PEPC from leaves of Amaranthus hypochondriacus was not stimulated, but was inhibited in the presence of ammonium. Our results demonstrate that ammonium ions stimulate PEPC by acting at the allosteric site. Ammonium ion being a component of plant metabolism could be an important regulator of PEPC, particularly in C4 plants.
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PMID:Ammonium ions stimulate in vitro the activity of phosphoenolpyruvate carboxylase from leaves of Amaranthus hypochondriacus, a C4 plant: evidence for allosteric activation. 795 Oct 51

The influence of pH on the in vitro activity and regulatory properties of Sorghum leaf C4 phosphoenolpyruvate carboxylase (PEPC) was investigated with respect to the phosphorylation status of the enzyme. In vitro protein phosphorylation was achieved using the catalytic subunit of a cAMP-dependent protein kinase (PKA) and a recombinant, immunopurified PEPC (0.9 mol of covalent Pi/mol PEPC subunit). Between pH 6.8 and 8, velocity and IC50 for L-malate increased for both the nonphosphorylated and the phosphorylated forms. With respect to the nonphosphorylated PEPC, the phospho-PEPC always gave high values for these kinetic parameters at the pH range investigated, especially between pH 7 and 7.3. The phosphorylation-induced stimulation of PEPC activity was four- to fivefold at pH 7.1 and approximately twofold at pH 7.3. The IC50 for L-malate showed a two- to threefold increase at pH 7.3, but varied less at pH 7.1 upon PEPC phosphorylation. Thus, phosphorylation of PEPC caused a predominant V effect or a mixed (V/IC50) effect at pH 7.1 or 7.3, respectively. This was also observed with the enzyme from desalted crude protein extracts from dark or light-adapted Sorghum leaves and leaf-derived mesophyll protoplasts illuminated in the presence of methylamine, a compound known to increase cytosolic pH (pHc). At pH 7.3, desensitization to L-malate of phospho-PEPC was due to an enhanced ability of PEP to compete with the inhibitor. The positive effector glucose-6P acted similarly to phosphorylation; however, a combination of both factors (glucose-6P and phosphorylation) led to a much larger increase in the IC50 for L-malate than that observed by a single factor.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The effect of pH on the covalent and metabolic control of C4 phosphoenolpyruvate carboxylase from Sorghum leaf. 798 87

Calcium-activated phosphoenolpyruvate carboxykinase from Escherichia coli is not inactivated by a number of sulfhydryl-directed reagents [5,5'-dithiobis(2-nitrobenzoate), iodoacetate, N-ethylmaleimide, N-(1-pyrenyl)maleimide or N-(iodoacetyl)-N'-(5-sulfo-1-naphthylethylenediamine)], unlike phosphoenolpyruvate carboxykinase from other organisms. On the other hand, the enzyme is rapidly inactivated by the arginyl-directed reagents 2,3-butanedione and 1-pyrenylglyoxal. The substrates, ADP plus PEP in the presence of Mn2+, protect the enzyme against inactivation by the diones. Quantitation of pyrenylglyoxal incorporation indicates that complete inactivation correlates with the binding of one inactivator molecule per mole of enzyme. Chemical modification by pyridoxal 5'-phosphate also produces inactivation of the enzyme, and the labeled protein shows a difference spectrum with a peak at 325 nm, characteristic of a pyridoxyl derivative of lysine. The inactivation by this reagent is also prevented by the substrates. Binding stoichiometries of 1.25 and 0.30 mol of reagent incorporated per mole of enzyme were found in the absence and presence of substrates, respectively. The results suggest the presence of functional arginyl and lysyl residues in or near the active site of the enzyme, and indicate lack of reactive functional sulfhydryl groups.
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PMID:Reactivity of cysteinyl, arginyl, and lysyl residues of Escherichia coli phosphoenolpyruvate carboxykinase against group-specific chemical reagents. 814 99

Whole leaves and guard-cell protoplasts of the C3 plant Vicia faba L. (broad bean) were separately extracted following a period of illumination or following a period of darkness. Kinetic parameters of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), Vmax and Km(PEP.Mg), were determined as a function of assay pH (7.0 or 8.1), the presence of 5 mM glucose-6-Pfree (Glc-6-P, an activator), and the presence of 5 mM malatefree (an inhibitor). On the basis of these parameters, guard-cell PEPC was distinguished from that of whole leaf, indicating either that guard cells contain a unique isoenzyme of PEPC or a different complement of isoenzymes or--and less likely--that the obligatorily different methodologies for the leaf (intact organ) and the guard-cell (protoplast) enzymes altered them specifically. The values of Vmax were relatively unchanged, regardless of assay conditions or tissue pretreatment. The values obtained for whole-leaf PEPC Vmax were restricted to a small range (52.4 +/- 5.9 (SD) to 64.4 +/- 4.8 (SD) mumol.g fresh mass-1.h-1; the high value coincided with the presence of Glc-6-P, and the low value was obtained in the presence of malate. Guard-cell PEPC Vmax was also restricted to a small range: 7.48 +/- 0.89 (SD) pmol.guard-cell pair-1.h-1 (pH 8.1, light, +Glc-6-P) to 5.79 +/- 0.60 (SD) pmol.guard-cell pair-1.h-1 (pH 7.0, dark, +malate). Depending on effectors, and particularly pH, large changes in Km(PEP.Mg) were calculated (whole-leaf PEPC: 0.03 to 3.84 mM; guard-cell PEPC: 0.06 to 3.43 mM).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Kinetic characterization of phosphoenolpyruvate carboxylase extracted from whole-leaf and from guard-cell protoplasts of Vicia faba L. (C3 plant) with respect to tissue pre-illumination. 815 Jun 61

Maize leaf phosphoenolpyruvate carboxylase was completely and irreversibly inactivated by treatment with micromolar concentrations of Woodward's reagent K (WRK) for about 1 min. The inactivation followed pseudo-first-order reaction kinetics. The order of reaction with respect to WRK showed that the reagent causes formation of reversible enzyme inhibitor complex before resulting in irreversible inactivation. The loss of activity was correlated to the modification of a single carboxyl group per subunit, even though the reagent reacted with 2 carboxyl groups per protomer. Substrate PEP and PEP+Mg2+ offered substantial protection against inactivation by WRK. The modified enzyme showed a characteristic absorbance at 346 nm due to carboxyl group modification. The modified enzyme exhibited altered surface charge as seen from the elution profile on FPLC Mono Q anion exchange column. The modified enzyme was desensitized to positive and negative effectors like glucose-6-phosphate and malate. Pretreatment of PEP carboxylase with diethylpyrocarbonate prevented WRK incorporation into the enzyme, suggesting that both histidine and carboxyl groups may be closely physically related. The carboxyl groups might be involved in metal binding during catalysis by the enzyme.
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PMID:Modification of maize phosphoenolpyruvate carboxylase by Woodward's reagent K. 825 Oct 65

The complete nucleotide (nt) sequence of the PEPCK gene encoding Trypanosoma cruzi phospho-enol-pyruvate carboxykinase (PEPCK; ATP dependent, EC 4.1.1.49) has been determined. The predicted primary sequence has 473 amino acids (aa) with a calculated molecular mass of 52.5 kDa. The ubiquitous spliced leader is present at nt position -60 from the AUG start codon in PEPCK mRNA; the coding region is followed by a long 3'-non-coding region of 777 nt. Northern and Southern blot analysis showed that the PEPCK mRNA is 2.7 kb long and that the PEPCK gene is polymorphic in T. cruzi, with more than one copy in the genome of the epimastigote form. Comparison of the available aa sequences of ATP(protozoa, yeast and bacteria)- and GTP(vertebrates, insects, helminths and fungi)-dependent PEPCKs showed that the former lack two characteristic, highly conserved regions present in the GTP-dependent enzymes: one is associated with the binding of PEP while the second is frequently labeled as 'catalytic' and contains a conserved Cys residue of unusual reactivity. On the other hand, two consensus sequences with conserved predicted secondary structure were identified in all PEPCKs, independent of their nt specificity; one of them is a divalent metal-binding site previously identified in pyruvate kinase by X-ray crystallographic studies.
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PMID:Cloning and characterization of the gene encoding ATP-dependent phospho-enol-pyruvate carboxykinase in Trypanosoma cruzi: comparison of primary and predicted secondary structure with host GTP-dependent enzyme. 829 43

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

We studied the transition metal ion requirements for activity and sulfhydryl group reactivity in phospho enol pyruvate carboxykinase (PEP-carboxykinase; ATP:oxaloacetate carboxylase (transphosphorylating), EC 4.1.1.49), a key enzyme in the energy metabolism of the protozan parasite Trypanosoma (Schizotrypanum) cruzi. As for other PEP-carboxykinases this enzyme has a strict requirement of transition metal ions for activity, even in the presence of excess Mg2+ ions for the carboxylation reaction; the order of effectiveness of these ions as enzyme activators was: Co2+ > Mn2+ > Cd2+ > Ni2+ >> Fe2+ > VO2+, while Zn2+ and Ca2+ had no activating effects. When we investigated the effect of the varying type or concentration of the transition metal ions on the kinetic parameters of the enzyme the results suggested that the stimulatory effects of the transition metal center were mostly associated with the activation of the relatively inert CO2 substrate. The inhibitory effects of 3-mercaptopicolinic acid (3MP) on the enzyme were found to depend on the transition metal ion activator: for the Mn(2+)-activated enzyme the inhibition was purely non-competitive (Kii = Kis) towards all substrates, while for the Co(2+)-activated enzyme the inhibitor was much less effective, produced a mixed-type inhibition and affected differentially the interaction of the enzyme with its substrates. The modification of a single, highly reactive, cysteine per enzyme molecule by 5,5'-dithiobis (2-nitro-benzoate) (DTNB) lead ton an almost complete inhibition of Mn(2+)-activated T. cruzi PEP-carboxykinase; however, in contrast with the results of previous studies in vertebrate and yeast enzymes, the substrate ADP slowed the chemical modification and enzyme inactivation but did not prevent it. PEP and HCO3- had no significant effect on the rate or extent of the enzyme inactivation. The kinetics of the enzyme inactivation by DTNB was also dependent on the transition metal activator, being much slower for the Co(2+)-activated enzyme than for its Mn(2+)-activated counterpart. When the bulkier but more hydrophobic reagent N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM) was used the enzyme was slowly and incompletely inactivated in the presence of Mn2+ and ADP afforded almost complete protection from inactivation; in the presence of Co2+ the enzyme was completely resistant to inactivation. Taken together, our results indicate that the parasite enzyme has a specific requirement of transition metal ions for activity and that they modulate the reactivity of a single, essential thiol group, different from the hyperreactive cysteines present in vertebrate or yeast enzymes.
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PMID:Trypanosoma cruzi phospho enol pyruvate carboxykinase (ATP-dependent): transition metal ion requirement for activity and sulfhydryl group reactivity. 854 43

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


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