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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 participation of lysine in the catalysis by avian liver phosphoenolpyruvate carboxykinase was studied by chemical modification and by a characterization of the modified enzyme. The rate of inactivation by 2,4-pentanedione is pseudo-first-order and linearly dependent on reagent concentration with a second-order rate constant of 0.36 +/- 0.025 M-1 min-1. Inactivation by pyridoxal 5'-phosphate of the reversible reaction catalyzed by phosphoenolpyruvate carboxykinase follows bimolecular kinetics with a second-order rate constant of 7700 +/- 860 M-1 min-1. A second-order rate constant of inactivation for the irreversible reaction catalyzed by the enzyme is 1434 +/- 110 M-1 min-1. Treatment of the enzyme with pyridoxal 5'-phosphate gives incorporation of 1 mol of pyridoxal 5'-phosphate per mole of enzyme or one lysine residue modified concomitant with 100% loss in activity. A stoichiometry of 1:1 is observed when either the reversible or the irreversible reactions catalyzed by the enzyme are monitored. A study of kobs vs pH suggests this active-site lysine has a pKa of 8.1 and a pH-independent rate constant of inactivation of 47,700 M-1 min-1. The phosphate-containing substrates IDP, ITP, and phosphoenolpyruvate offer almost complete protection against inactivation by pyridoxal 5'-phosphate. Modified, inactive enzyme exhibits little change in Mn2+ binding as shown by EPR. Proton relaxation rate measurements suggest that pyridoxal 5'-phosphate modification alters binding of the phosphate-containing substrates. 31P NMR relaxation rate measurements show altered binding of the substrates in the ternary enzyme.Mn2+.substrate complex. Circular dichroism studies show little change in secondary structure of pyridoxal 5'-phosphate modified phosphoenolpyruvate carboxykinase. These results indicate that avian liver phosphoenolpyruvate carboxykinase has one reactive lysine at the active site and it is involved in the binding and activation of the phosphate-containing substrates.
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PMID:An active-site lysine in avian liver phosphoenolpyruvate carboxykinase. 190 75

The modification of avian phosphoenolpyruvate carboxykinase by a variety of sulfhydryl reagents leads to inhibition. The inhibition is related to the loss of 1 highly reactive cysteine residue of the 13 cysteines present in the enzyme. Inhibition by reagents which yield a mixed disulfide was rapidly reversed by thiols. Reagents specific for vicinal sulfhydryl configurations were not potent inhibitors. The cysteine-modified enzyme continues to bind Mn2+ with the same stoichiometry and dissociation constant as the native enzyme. All of the substrates also bind to thiol-modified inactive enzyme. The modification of the reactive cysteine with the spin-labeled iodoacetate derivative leads to inactive enzyme with spin label stoichiometrically incorporated. The EPR spectrum showed an immobilized spin label on the enzyme. EPR studies of the perturbation of the phosphoenolpyruvate carboxykinase-bound spin label by bound Mn2+ showed a dipolar interaction between the two spins, estimated to be 10 A apart. The perturbation of the 1/T1 and 1/T2 values of the 31P resonances of ITP by spin-labeled enzyme indicates that this portion of the nucleotide binds 8-10 A from the spin label. These results indicate that the reactive cysteine is close to but not at the active site of the enzyme. The thiol group must be free and in its reduced form for the enzyme to be active. Perhaps modification of this group prevents conformational change(s) upon ligand binding necessary for the catalytic process.
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PMID:A reactive cysteine in avian liver phosphoenolpyruvate carboxykinase. 254 99

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

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) is activated by Cr2+ as the sole activator under anaerobic conditions. PEPCK was modified with Cr3+, starting with either Cr2+ or Cr3+. Cr3+ has the distinct advantage of being a paramagnetic cation that could serve as a paramagnetic probe. Activators Mn2+, Mg2+, and Co2+ protect against Cr3+ incorporation. EPR, CD, and fluorescence studies indicate that Cr3+ was incorporated into the cation binding site of PEPCK. The water proton relaxation rate (PRR) and fluorescence binding studies showed that Cr3+(n1)-PEPCK forms enzyme-substrate complexes similar to those observed for the Mn2+(n1)-PEPCK complex (n1 represents the metal "enzyme binding site" as opposed to the metal "nucleotide binding site"). Cr3+(n1)-PEPCK requires an additional divalent cation for activity, an indication of two metal sites on PEPCK. Cr3+(n1)-PEPCK retains 15% residual activity as compared to unmodified PEPCK and demonstrates normal Michaelis-Menten kinetics. This is the first report of an active Cr3+-modified enzyme complex.
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PMID:Chromium(III) modification of the first metal binding site of phosphoenolpyruvate carboxykinase. 960

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

Embryo axes isolated from germinating lupine seeds were cultivated in vitro for 24-96 h over media containing either 60 mmol/L sucrose or no sucrose. Ultrastructural studies showed that large vacuoles were accumulating in a central region of primary parenchyma cells in sucrose starved lupine embryo axes, whereas cytoplasm along with organelles were forced to a periphery of the cells. We suggest that the autolysis of cytoplasmic proteins contributes to the accumulation of the vacuoles and this suggestion is consistent with the results of the characterisation of protein content. The level of cytosolic proteins was reduced by 50% and the activity of cytosolic marker enzyme, PEP carboxylase, was reduced by 46% in starved embryos as compared to control. The mitochondria from starved tissues were not degraded. The level of mitochondrial proteins was reduced by only 10% and the activity of mitochondrial NAD-isocitrate dehydrogenase decreased by 8% as a result of starvation. As demonstrated by the results of Percoll density gradient centrifugation, sucrose starvation caused an increase of 49% in many of the higher density mitochondria fractions, whereas many of the lower density mitochondria fractions were decreased by 33%. The samples of mitochondria from starved embryo axes were determined to have higher respiration activity in the presence of glutamate and malate as compared to control samples. EPR-based analyses of free radicals showed the presence of free radicals with a signal at g = 2.0060 in embryo axes. The level of the radical was two times higher in sucrose-starved embryo axes than in control (the level of this radical increased in senescing plant tissues as well). The results of EPR-based quantitation of Mn2+ ions revealed that the level was a few times higher in starved material than in control. Starved embryo axes, however, do possess a number of adaptive mechanisms protecting them from oxidative damage. Densitometric analyses of gels revealed an increase in the activity of SOD in sugar-starved embryos, whereas CAT and POX activities were lower in axes grown without sucrose as compared to control. Superoxide dismutase, catalase and peroxidase zymogram analyses showed that synthesis of new isoforms was not induced by sugar starvation. An accumulation of phytoferritin was found in plastids of sucrose starved embryos. These results are discussed in relation to the metabolic changes observed in senescing plant tissues.
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PMID:Metabolic and ultrastructural responses of lupine embryo axes to sugar starvation. 1274 88