Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:4.1.1.49 (phosphoenolpyruvate carboxykinase)
4,654 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Carbon isotope effects for the carbon atom arising from bicarbonate have been measured for the phosphoenolpyruvate carboxylase from maize. At pH 7.5, 25 degrees C, the isotope effect is K12/k13 = 1.0029 +/- 0.0005 in the presence of Mg2+. The isotope effect decreases with increasing pH, reaching a value of 0.9973 at pH 10.0. All these isotope effects are relative to HCO3(-) taken as the starting state. If CO2 is considered the starting state, the isotope effects are all inverse. These values suggest that the carboxylation of phosphoenolpyruvate occurs by way of a stepwise mechanism involving an enzyme-bound carboxyphosphate intermediate, with formation of the intermediate being the primary rate-determining step. Steady-state kinetics reveal that Vmax is independent of pH over the range pH 7.5-10.0 Vmax/Km (phosphoenolpyruvate) is bell shaped in the same interval. Two pKa values near 7 are observed; the first is attributed to ionization of the phosphate group of phosphoenolpyruvate and the second to an unidentified group on the enzyme. Activity of the enzyme also depends on protonation of a group on the enzyme with a pKa near 10. Several metal ions were tested as activators of phosphoenolpyruvate carboxylase. Under saturating conditions, Mg2+ and Mn2+ show equal activity but different carbon isotope effects. Co2+ has about half the activity of Mg2+ and shows an inverse carbon isotope effect.
...
PMID:Kinetic and isotope effect studies of maize phosphoenolpyruvate carboxylase. 731 83

ATP-dependent phospho enol pyruvate carboxykinase (EC 4.1.1.49; PEPCK, ATP) was purified from glycosomes of cultured procyclic Trypanosoma brucei to electrophoretic homogeneity. The purified enzyme exhibited a mean specific activity of 83 units mg-1, as measured in the carboxylation direction at 30 degrees C. A similar activity was obtained for the decarboxylation reaction. The enzyme was shown to be a homodimer in solution with a subunit molecular mass of 59 kDa. Amino acid sequence analysis suggested that the PEPCK (ATP) is identical to the trypanosomal protein p60, the sequence of which was previously predicted from the corresponding nucleotide sequence by other investigators. The basic nature of the enzyme was indicated by a high isoelectric point (pH 8.9). The enzyme was found to be strictly dependent on adenosine nucleotides for activity, as well as on the presence of Mn2+. Mg2+ was found to be ineffective as activator of the trypanosomal enzyme, but a combination of subsaturating (< or = 300 microM) concentrations of Mn2+ and high concentrations of Mg2+ caused a synergistic effect on the carboxylation activity, indicating a dual cation requirement. Mn2+ is necessary to activate the enzyme and Mn2+ or Mg2+ most likely forms the cation-nucleotide complex as the active form of the substrate. Relatively high (5 mM) levels of ATP were required to produce a significant inhibition of the carboxylation reaction. Quinolinic acid, a structural analogue of oxaloacetate, completely inhibited the decarboxylation reaction at a 1 mM concentration. The apparent Michaelis constants of the enzyme were 490 microM for PEP, 37 microM for oxaloacetate, 40 microM for ADP, 10.3 microM for ATP, 970 microM for Mn2+ and 26 mM for HCO3-. Endogenous substrate concentrations were found to be 327 nmol PEP, 1486 nmol ADP, 4200 nmol ATP and 11.5 nmol Mn2+ (ml cell volume)-1. Our kinetic data suggest that under physiological conditions PEPCK (ATP) in T. brucei is bidirectional and that its activity is regulated primarily by mass action. The physiological relevance of the enzyme in procyclic T. brucei is discussed.
...
PMID:Purification and characterization of phospho enol pyruvate carboxykinase from Trypanosoma brucei. 776 79

Two members of the ATP-dependent class of phospho enol pyruvate (PEP) carboxykinases (Saccharomyces cerevisiae and Escherichia coli PEP carboxykinase), and one member of the GTP-dependent class (the cytosolic rat liver enzyme) have been comparatively analyzed by taking advantage of their intrinsic fluorescence. The S. cerevisiae and the rat liver enzymes show intrinsic fluorescence with a maximum emission characteristic of moderately buried tryptophan residues, while the E. coli carboxykinase shows somewhat more average exposure for these fluorophores. The fluorescence of the three proteins was similarly quenched by the polar compound acrylamide, but differences were observed for the ionic quencher iodide. For the ATP-dependent enzymes, these last experiments indicate more exposure to the aqueous media of the tryptophan population of the E. coli than of the S. cerevisiae enzyme. The effect of nucleotides on the emission intensities and quenching efficiencies revealed substrate-induced conformational changes in the E. coli and cytosolic rat liver PEP carboxykinases. The addition of Mn2+ or of the adenosine nucleotides in the presence of Mg2+ induced an enhancement in the fluorescence of the E. coli enzyme. The addition of guanosine or inosine nucleotides to the rat liver enzyme quenched its fluorescence. From the ligand-induced fluorescence changes, dissociation constants of 40 +/- 6 microM, 10 +/- 0.8 microM, and 15 +/- 1 microM were obtained for Mn2+, MgATP and MgADP binding to the E. coli enzyme, respectively. For the cytosolic rat liver PEP carboxykinase, the respective values for GDP, IDP and ITP binding are 6 +/- 0.5 microM, 6.7 +/- 0.4 microM and 10.1 +/- 1.7 microM. A comparison of the dissociation constants obtained in this work with those reported for other PEP carboxykinases is presented.
...
PMID:Comparative steady-state fluorescence studies of cytosolic rat liver (GTP), Saccharomyces cerevisiae (ATP) and Escherichia coli (ATP) phospho enol pyruvate carboxykinases. 844 84

The presence of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31), an enzyme at the branchpoint of glycolysis and the Krebs cycle was detected in the Filaria Molinema dessetae. This enzyme has not previously been identified in Helminths, which have so far been found to only possess a phosphoenolpyruvate carboxykinase (EC 4.1.1.32). This enzyme had a level of activity comparable to that of pyruvate kinase, and was relatively less active than enzymes such as malate dehydrogenase or lactate dehydrogenase. We propose here a method of purification of M. dessetae PEP-carboxylase. When purified to electrophoretic homogeneity, the enzyme had a molecular weight of 64 kDa. Kinetic studies indicated that the carboxylation reaction had an optimal pH of 5.8. The enzyme was inhibited by cations such as Fe2+, Zn2+, Cd2+, Cu2+ but required the presence of Mg2+ or Mn2+. The enzyme was thermostable. The apparent Km value of 2.38 mmol for phosphoenolpyruvate for the carboxylation reaction was higher than previously reported values. The Km value for KHCO3 was found to be 1.6 mmol. PEP-carboxylase did not catalyse the reverse reaction.
...
PMID:Purification and properties of phosphoenolpyruvate carboxylase from Molinema dessetae (Nematoda: Filarioidea). 847 1

A cytosolic cell-free system prepared from rat liver was used to study the effect of bivalent cations on the activity of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK). Steady-state concentrations of oxaloacetate in the range 5-50 microM were generated from increasing concentrations of malate+fumarate (10:1); 2 mM ITP and 3 mM Mg2+ were added as cofactors. Micromolar concentrations of Mn2+, Fe2+ and, to a lesser extent, of Zn2+ and Co2+ were shown to stimulate PEPCK activity. Vmax. (mumol/min per g of liver) increased from 0.67 to 1.68 on addition of 5 microM Fe2+ and to 2.34 with 2 microM Mn2+, whereas no significant effect on the Km for oxaloacetate was observed. The apparent K(a) values (total) were 0.62 microM for Mn2+, 1.48 microM for Zn2+, 1.92 microM for Co2+ and 3.37 microM for Fe2+, being 2-8-fold lower than the corresponding published values. Variations of the free Mn2+ concentration were obtained (a) by increasing the Mn2+ concentration (i.e. activation curve) and (b) by simultaneous addition of Mn2+ and increasing concentrations of the chelating agent EGTA (i.e. inactivation curve). Different results were obtained for the activation and inactivation curves. The inactivation curve showed that PEPCK activity was almost unaffected by variations of the free Mn2+ concentration over the range 0.05-0.15 microM. Under comparable experimental conditions, rat liver arginase (another Mn(2+)-dependent enzyme) was completely inactivated. From kinetic evidence, the existence of two distinct molecular forms of cytosolic rat liver PEPCK with different Mn2+ affinities is postulated. Considering the high affinity of PEPCK for Mn2+ and its relative insensitivity to changes in the free Mn2+ concentration, it seems rather unlikely that changes in the free cation concentration play a major role in regulating PEPCK activity in vivo.
...
PMID:A physiological role of Mn2+ in the regulation of cytosolic phosphoenolpyruvate carboxykinase from rat liver is unlikely. 850 71

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.
...
PMID:Trypanosoma cruzi phospho enol pyruvate carboxykinase (ATP-dependent): transition metal ion requirement for activity and sulfhydryl group reactivity. 854 43

Phosphoenolpyruvate (PEP) carboxykinase was purified 42-fold with a 25% yield from cell extracts of Ruminococcus flavefaciens by ammonium sulfate precipitation, preparative isoelectric focusing, and removal of carrier ampholytes by chromatography. The enzyme had a subunit molecular mass of approximately 66.3 kDa (determined by mass spectrometry), but was retained by a filter having a 100-kDa nominal molecular mass cutoff. Optimal activity required activation of the enzyme by Mn2+ and stabilization of the nucleotide substrate by Mg2+. GDP was a more effective phosphoryl acceptor than ADP, while IDP was not utilized. Under optimal conditions the measured activity in the direction of PEP carboxylation was 17.2 micromol min-1 (mg enzyme)-1. The apparent Km values for PEP (0.3 mM) and GDP (2.0 mM) were 9- and 14-fold lower than the apparent Km values for the substrates of the back reaction (oxaloacetate and GTP, respectively). The data are consistent with the involvement of PEP carboxykinase as the primary carboxylation enzyme in the fermentation of cellulose to succinate by this bacterium.
...
PMID:Purification and characterization of phosphoenolpyruvate carboxykinase from the anaerobic ruminal bacterium Ruminococcus flavefaciens. 909 26

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.
...
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.
...
PMID:Affinity cleavage at the metal-binding site of phosphoenolpyruvate carboxykinase. 939 80

We discovered that Methanobacterium thermoautotrophicum strain DeltaH possessed pyruvate carboxylase (PYC), and this biotin prototroph required exogenously supplied biotin to exhibit detectable amounts of PYC activity. The enzyme was highly labile and was stabilized by 10% inositol in buffers to an extent that allowed purification to homogeneity and characterization. The purified enzyme was absolutely dependent on ATP, Mg2+ (or Mn2+ or Co2+), pyruvate, and bicarbonate for activity; phosphoenolpyruvate could not replace pyruvate, and acetyl-CoA was not required. The enzyme was inhibited by ADP and alpha-ketoglutarate but not by aspartate or glutamate. ATP was inhibitory at high concentrations. The enzyme, unlike other PYCs, exhibited nonlinear kinetics with respect to bicarbonate and was inhibited by excess Mg2+, Mn2+, or Co2+. The 540-kDa enzyme of A4B4 composition contained a non-biotinylated 52-kDa subunit (PYCA) and a 75-kDa biotinylated subunit (PYCB). The pycB gene was probably monocistronic and followed by a putative gene of a DNA-binding protein on the opposite strand. The pycA was about 727 kilobase pairs away from pycB on the chromosome and was probably co-transcribed with the biotin ligase gene (birA). PYCA and PYCB showed substantial sequence identities (33-62%) to, respectively, the biotin carboxylase and biotin carboxyl carrier + carboxyltransferase domains or subunits of known biotin-dependent carboxylases/decarboxylases. We discovered that PYCB and probably the equivalent domains or subunits of all biotin-dependent carboxylases harbored the serine/threonine dehydratase types of pyridoxal-phosphate attachment site. Our results and the existence of an alternative oxaloacetate synthesizing enzyme phosphoenolpyruvate carboxylase in M. thermoautotrophicum strain DeltaH (Kenealy, W. R., and Zeikus, J. G. (1982) FEMS Microbiol. Lett. 14, 7-10) raise several questions for future investigations.
...
PMID:Purification, regulation, and molecular and biochemical characterization of pyruvate carboxylase from Methanobacterium thermoautotrophicum strain deltaH. 947 69


<< Previous 1 2 3 4 5 6 Next >>

---