Gene/Protein Disease Symptom Drug Enzyme Compound
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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 interactions of nucleotide substrates with the enzyme phosphoenolpyruvate carboxykinase and its Mn2+ complex were investigated by several methods. Direct binding shows the formation of stoichiometric complexes. The presence of Mn2+ increases the affinity of the enzyme for nucleotide. A higher affinity for GTP (Kd less than 2 microM) than for GDP (Kd = 15 microM) was determined. Solvent proton relaxation rate studies indicate no substantial difference in titration curves for free nucleotide or for Mg-nucleotide to the enzyme-Mn complex. The effect of Mn2+ on the 31P relaxation rates of IDP and of ITP in the binary Mn-nucleotide complex indicates the formation of direct coordination complexes. The distances of the alpha- and beta-31P of IDP to Mn2+ are identical (3.5 A). The Mn2+ distance to the beta- and gamma-31P of ITP is also identical (3.7 A) and is 0.2 A further from the alpha-phosphorus. In the presence of P-enolpyruvate carboxykinase, the effect of Mn2+ on the 31P relaxation rates was measured at 40.5 MHz and at 121.5 MHz. The dipolar correlation time was calculated to be 0.6-5.4 ns, depending upon assumptions made. The Mn2+ to phosphorus distances indicate the nucleotide substrates form a second sphere complex to the bound Mn2+. From 1/T2 measurements, electron delocalization from Mn2+ to the phosphorus atoms is indicated; this effect occurs although direct coordination does not take place. The exchange rate of GTP from the enzyme-Mn complex (koff = 4 X 10(4) s-1) is rapid compared to kcat with a lower energy of activation (9.2 kcal/mol) than for catalytic turnover.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Phosphorus-31 nuclear relaxation rate studies of the nucleotides on phosphoenolpyruvate carboxykinase. 652 66

The inhibition of chicken liver phosphoenolpyruvate carboxykinase by 3-mercaptopicolinic acid (3-MP) has been investigated. Kinetic studies show 3-MP to be a noncompetitive inhibitor relative to all substrates and to the activator, Mn2+. EPR studies demonstrate that Mn2+ binding to the enzyme is unaffected by 3-MP. Proton relaxation rate studies demonstrate that 3-MP binds to the binary E X Mn complex with a KD of 0.5 X 10(-6) M and gives a ternary enhancement of 8.0. Additional proton relaxation rate studies detected formation of the quaternary complexes E X Mn X IDP X 3-MP, E X Mn X ITP X 3-MP, and E X Mn X CO2 X 3-MP. High resolution 1H nuclear relaxation rate studies suggest that 3-MP binds in close proximity to the activator cation, Mn2+, but not in the first coordination sphere. Active site models suggest that the 3-MP-binding site may partially overlap the phosphoenolpyruvate-binding site. The NMR studies, which detected formation of the quaternary E X Mn X 3-MP X phosphoenolpyruvate complex, also demonstrated that the binding of one of these ligands affects the interactions of the other ligand with E X Mn. Calorimetric studies of the E X Mn complex demonstrated that 3-MP causes an increase in the transition temperature midpoint without an increase in enthalpy. These results indicate that 3-MP causes a conformational change in the enzyme but does not increase the thermostability of the ternary complex. The experiments reported herein suggest that inhibition by 3-MP is due to specific and reversible binding within the active site of phosphoenolpyruvate carboxykinase.
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PMID:3-Mercaptopicolinate. A reversible active site inhibitor of avian liver phosphoenolpyruvate carboxykinase. 661 35

The activation of the avian liver phosphoenolpyruvate carboxykinase catalyzed reversible decarboxylation of oxalacetate by Mn2+ has been studied. The Mn2+ facilitates the interaction of oxalacetate to the enzyme. At saturating ITP and oxalacetate concentrations, Mn2+ has a Km = 2 microM. The cation Mg2+ can substitute for Mn2+ with 50% Vmax in the oxalacetate decarboxylation and 2% Vmax in the oxalacetate formation. The Km for Mg2+ is 3 orders of magnitude greater than the Km of Mn2+, however. Of the other cations tested (Ca2+, Zn2+, Fe2+, and Cd2+), Co2+ was the only other cation found to activate the enzyme in both directions. In the presence of Mg2+, the enzyme is extremely sensitive to trace metal contaminants which can cause activation. At 1 mM Mg2+, 20 microM Mn2+ causes a 15-fold activation of activity. The apparent Km for Mn2+ (2 microM) at high concentrations of Mg2+ is the identical value calculated for free Mn2+. In a mixed metal (Mg2+ and Mn2+) assay, the Km values for phosphoenolpyruvate and for oxalacetate are independent of the concentration of Mg2+ but decrease upon an increase in Mn2+. The kinetic results demonstrate two roles for the divalent cations for activity. The cation forms a metal . nucleotide complex which serves as the substrate. The results indicate that MgITP is a better substrate than MnITP, but MnIDP is a better substrate than MgIDP. The cation also binds to the enzyme to form an enzyme . metal complex which is the active form of the enzyme. This cation functions to aid in the interaction of either oxalacetate or phosphoenolpyruvate to the enzyme. The activation by micromolar amounts of Mn2+ at millimolar concentrations of Mg2+ suggests that this enzyme, and thus the pathway of gluconeogenesis, can be modulated by changes in concentration of Mn2+ within mitochondria.
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PMID:The role of cations in avian liver phosphoenolpyruvate carboxykinase catalysis. Activation and regulation. 679 77

The enzyme phosphoenolpyruvate carboxykinase has been purified from chicken liver mitochondria. This purification includes a pseudo-affinity column step utilizing Sepharose 4B-blue dextran which binds the enzyme. The enzyme elutes with ITP to yield protein which is greater than 98% pure. The enzyme has Mr = 75,400 +/- 200 estimated by high speed sedimentation equilibrium and 70,500 +/- 500 estimated by reduced sodium dodecyl sulfate-polyacrylamide gels. The enzyme is abnormally retarded on molecular exclusion resins yielding low apparent molecular weight values. The amino acid analysis indicates that the enzyme has a high proline content and a high tryptophan content and contains 9 mol of cysteine/mol of enzyme. No disulfide bonds were detected. The extinction coefficient (epsilon 1% 280 = 16.5 +/- 0.1) reflects the high tryptophan content. The Svedberg coefficient (s20,w = 4.63 +/- 0.03 S) is consistent with a globular protein of Mr = 70,500-75,400. The activation of the enzyme was investigated by steady state kinetics. The carboxylation reaction has an activation energy of 17.6 kcal/mol. There is no requirement of a monovalent cation for activity. A thiol is necessary for maximal activity, although apparently not to reduce disulfide bonds within the enzyme. Incubation with dithiothreitol stabilizes enzymatic activity but beta-mercaptoethanol facilitates loss of activity. The kinetics of activation by Mn2+ was performed. The Ks value for phosphoenolpyruvate (300 microM) decreases to an apparent Km of 67 microM with increasing concentrations of Mn2+. The concentration of Mn2+ does not affect the interaction of HCO-3 with the enzyme, however. Analysis of data in terms of free IDP indicates that increasing Mn2+ decreases the Km of IDP but analysis as MnIDP indicates the Km,app of MnIDP is independent of the Mn2+ concentration. The enzyme interacts with Mn2+ with a KA = 67 microM and the Km,app decreases to a value of 8 microM with saturating substrates. The substrate analogue (Z)-3-fluorophosphoenolpyruvate is a good substrate for the reaction (Km = 30 microM) with 27% Vmax compared to P-enolpyruvate (Km = 180 microM). Except for 3-bromophosphoenolpyruvate, other analogues have shown weak competitive or noncompetitive inhibition. Potential analogues of oxalacetate (succinate, citrate, isocitrate, malate, and alpha-ketoglutarate) all elicit weak (greater than 15 mM) inhibition.
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PMID:The purification, characterization, and activation of phosphoenolpyruvate carboxykinase from chicken liver mitochondria. 706 3

Streptonigrin, an antibiotic with antineoplastic activity, inhibited rat liver phosphoenolpyruvate carboxykinase with an I50 of 0.3 microM when excess FeCl2 was present. No inhibition occurred in the absence of added metal ion. Inhibition was partial and noncompetitive versus ITP and oxalacetic acid. The enzyme was more susceptible to inhibition by streptonigrin in the absence of substrates. Fe2+ supported inhibition by streptonigrin to a greater extent than did Fe3+, while Mn2+ activated the enzyme in the presence of streptonigrin. For maximum inhibition, at least a 3-fold molar excess of iron over streptonigrin was required. The methyl ester of streptonigrin was also an inhibitor (I50 = 4 microM) while the fragment containing the C and D rings was not, indicating that inhibition did not depend solely on the presence of the picolinic acid moiety. When oxalacetate synthesis was measured, streptonigrin plus iron had no more effect on enzymatic activity than iron alone, and Mn2+ was capable of stimulating the streptonigrin-Fe2+ inhibited enzyme.
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PMID:Inhibition of phosphoenolpyruvate carboxykinase by streptonigrin. 708 67

A method is described for the purification of the enzyme phosphoenolpyruvate carboxykinase (PEPCK) from the cestode Hymenolepis diminuta. When purified to electrophoretic homogeneity, the enzyme had a molecular weight of 70,600 and an isoelectric point of 7.5. Kinetic studies indicated that the pH 5.6 was optimal for the carboxylation reaction and that Mn++ was the preferred divalent cation; there was no activity of the enzyme in the presence of Mg++. Apparent Km values for the carboxylation reaction were determined; those for GDP (20.6 muM) and PEP (38.9 muM) were lower than the values previously reported. GTP, GMP, ITP, IMP, fumarate, succinate and alpha-ketoglutarate were found to be competitive inhibitors and their Ki values determined.
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PMID:Purification and properties of phosphoenolpyruvate carboxykinase from Hymenolepis diminuta (Cestoda). 732 56

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.
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PMID:A physiological role of Mn2+ in the regulation of cytosolic phosphoenolpyruvate carboxykinase from rat liver is unlikely. 850 71

The phosphoenolpyruvate carboxykinase (PEPCK) from Vibrio costicola catalyzed a 14CO2-oxaloacetate exchange reaction with an unusual nucleotide specificity. ATP gave the higher apparent catalytic efficiency (Vmax/Km, 6.78), followed by GTP (1.30), CTP (0.87) and ITP (0.66). Maximal activity required a divalent cation; CdCl2 and MgCl2 synergistically activated the enzyme, when added in the presence of MnCl2. The sigmoidal saturation curve for MnCl2 (apparent n 2.11) was converted into a hyperbola by 0.01 mM CdCl2 (apparent n 1). The results suggest a double role of the divalent cation in the reaction mechanism, namely as part of the MeATP2- substrate and as free Me2+. Mn2+ would be the best for the first, and Cd2+ for the second role. Preincubation with 0.01 mM CdCl2 increased the activity of the enzyme assayed with MgATP2- through an increase in Vmax; addition of CdCl2 to the reaction mixture elicited further activation, through a 17-fold decrease in the apparent Km for MgATP2-. These results, together with the biphasic curve of activation by CdCl2 when used alone, suggest the existence of two different sites for free Cd2+ on the enzyme.
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PMID:Effects of divalent cations and nucleotides on the 14CO2-oxaloacetate exchange catalyzed by the phosphoenol pyruvate carboxykinase from the moderate halophile, Vibrio costicola. 853 94

The enzyme phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the reversible conversion of oxalacetate and GTP to phosphoenolpyruvate (PEP), GDP, and CO2. PEPCK from higher organisms is a monomer, specifically requires GTP or ITP, and uses Mn2+ as the activating cation. Currently, there is no crystal structure of GTP-utilizing PEPCKs. The conformation of the bound nucleotide was determined from transferred nuclear Overhauser effects (trnOe) experiments to determine internuclear proton distances. At 600 MHz in the presence of PEPCK, nOe effects were observed between nucleotide protons. Internuclear distances were calculated from the initial rate of the nOe buildup. These distance constraints were used in energy minimization calculations to determine the conformation of PEPCK-bound GTP. The bound nucleotide has the base oriented anti to the C2'-endo(2E) ribose ring conformation. Relaxation rate studies indicate that there is an additional relaxation effect on the C1' proton upon nucleotide binding to PEPCK. Nucleotide binding to PEPCK-Mn2+ was studied by 1H relaxation rate studies, but results were complicated by long dipole-dipole distances and the presence of competing complexes. Modification of PEPCK by iodoacetamido-TEMPO leads to an inactive enzyme that is spin-labeled at cys273. The interaction of TEMPO-PEPCK with GTP allows for the measurement of nuclear distances between GTP and the spin label. The results suggest that cys273 lies near the ribose ring of the bound nucleotide, but it is too far to be implicated in direct hydrogen bonding interactions consistent with previous results [Makinen, A. L., and Nowak, T. J. Biol. Chem. (1989) 264, 12148], suggesting that cys273 does not actively participate in catalysis. Modification of PEPCK with several cysteine specific modifying agents causes no change in the ability of the enzyme to bind nucleotide as monitored by fluorescence quenching. A correlation between the size of the modifying agent and the maximal observed quenching upon saturation of the enzyme with nucleotide is observed. This suggests a mechanism for inactivation of PEPCK by cysteine modification due to inhibition of a dynamic motion that may occur upon nucleotide binding.
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PMID:Structural investigation of the binding of nucleotide to phosphoenolpyruvate carboxykinase by NMR. 1155 Dec

Phosphoenolpyruvate partially inhibits the accumulation of Ca(2+) in isolated mung bean (Phaseolus aureus Roxb.) mitochondria. Succinate-supported Ca(2+) uptake is twice as sensitive to phosphoenolpyruvate inhibition as is NADH- or malate/pyruvate-supported Ca(2+) uptake. Pyruvate, atractylate, and ATP, but not ITP, reverse the phosphoenolpyruvate-induced inhibition. Oxaloacetic acid inhibits succinate-supported Ca(2+) uptake completely while partially inhibiting NADH-supported Ca(2+) uptake. The oxaloacetate inhibition of NADH-supported Ca(2+) uptake is greater than that produced by phosphoenolpyruvate. It is suggested that inhibition of Ca(2+) uptake is due to the conversion of phosphoenolpyruvate into oxaloacetate via phosphoenolpyruvate carboxykinase, with oxaloacetate responsible for the actual inhibition of Ca(2+) uptake.
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PMID:Effect of phosphoenolpyruvate and oxaloacetate on ca uptake by isolated mung bean mitochondria. 1665


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