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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)

A method has been developed for rapidly preparing bundle sheath cell strands from Urochloa panicoides, a phosphoenolpyruvate (PEP) carboxykinase-type C4 plant. These cells catalyzed both HCO3(-)- and oxaloacetate-dependent oxygen evolution; oxaloacetate-dependent oxygen evolution was stimulated by ATP. For this activity oxaloacetate could be replaced by aspartate plus 2-oxoglutarate. Both oxaloacetate- and aspartate plus 2-oxoglutarate-dependent oxygen evolution were accompanied by PEP production and both were inhibited by 3-mercaptopicolinic acid, an inhibitor of PEP carboxykinase. The ATP requirement for oxaloacetate- and aspartate plus 2-oxoglutarate-dependent oxygen evolution could be replaced by ADP plus malate. The increased oxygen evolution observed when malate plus ADP was added with oxaloacetate was accompanied by pyruvate production. These results are consistent with oxaloacetate being decarboxylated via PEP carboxykinase. We suggest that the ATP required for oxaloacetate decarboxylation via PEP carboxykinase may be derived by phosphorylation coupled to malate oxidation in mitochondria. These bundle sheath cells apparently contain diffusion paths for the rapid transfer of compounds as large as adenine nucleotides.
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PMID:Photosynthesis in phosphoenolpyruvate carboxykinase-type C4 plants: photosynthetic activities of isolated bundle sheath cells from Urochloa panicoides. 312 45

Previous attempts to account for the labelling in vivo of liver metabolites associated with the citrate cycle and gluconeogenesis have foundered because proper allowance was not made for the heterogeneity of the liver. In the basal state (anaesthetized after 24h starvation) this heterogeneity is minimal, and we show that labelling by [14C]bicarbonate can be interpreted unambiguously. [14C]Bicarbonate was infused to an isotopic steady state, and measurements were made of specific radioactivities of blood bicarbonate, alanine, glycerol and lactate, of liver alanine and lactate, and of individual carbon atoms in blood glucose and liver aspartate, citrate and malate. (Existing methods for several of these measurements were extensively modified.) The results were combined with published rates of gluconeogenesis, uptake of gluconeogenic precursors by the liver, and citrate-cycle flux, all measured under similar conditions, and with estimates of other rates made from published data. To interpret the results, three ancillary measurements were made: the rate of CO2 exchange by phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) under conditions that simulated those in vivo; the 14C isotope effect in the pyruvate carboxylase (EC 6.4.1.1) reaction (14C/12C = 0.992 +/- 0.008; S.E.M., n = 8); the ratio of labelling by [2-14C]- to that by [1-14C]-pyruvate of liver glutamate 1.5 min after injection. This ratio, 3.38, is a measure of the disequilibrium in the mitochondria between malate and oxaloacetate. The data were analysed with due regard to experimental variance, uncertainties in values of fluxes measured in vitro, hepatic heterogeneity and renal glucose output. The following conclusions were reached. The results could not be explained if CO2 fixation was confined to pyruvate carboxylase and there was only one, well-mixed, pool of oxaloacetate in the mitochondria. Addition of the other carboxylation reactions, those of PEPCK, isocitrate dehydrogenase (EC 1.1.1.42) and malic enzyme (EC 1.1.1.40), was not enough. Incomplete mixing of mitochondrial oxaloacetate had to be assumed, i.e. that there was metabolic channelling of oxaloacetate formed from pyruvate towards gluconeogenesis. There was some evidence that malate exchange across the mitochondrial membrane might also be channelled, with incomplete mixing with that in the citrate cycle. Calculated rates of exchange of CO2 by PEPCK were in agreement with those measured in vitro, with little or no activation by Fe2+ ions.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:[14C]bicarbonate fixation into glucose and other metabolites in the liver of the starved rat under halothane anaesthesia. Metabolic channelling of mitochondrial oxaloacetate. 392 30

A crude preparation of PEP carboxylase (EC 4.1.1.31) from the yellow lupin roots exhibits the pH optimum of activity within the range of 7.4-8.6 and the temperature optimum at 32 - 40 degrees C. Its Km for PEP is 0.1 mM, and Km for HCO3- is 0.7 mM. The affinity of the enzyme towards Mg2+ diminishes with the metal ion concentration. At the concentration of Mg2+ below 0.5 mM Km for Mg2+ is 0.07 mM and at the Mg2+ concentration over 1.5 mM it rises to 0.47 mM. The Hill coefficients are 0.37 and 0.88, respectively. Among several compounds affecting the PEP carboxylase activity, such as organic acids, amino acids, and sugar phosphates, at physiological pH (7.0 and 7.8), malate shows the strongest inhibition of a competitive character, its Ki being 2 mM. Also acidic amino acids strongly inhibit the enzyme activity, aspartate being more effective than glutamate. Glucose 6-phosphate and fructose 1,6-diphosphate markedly activate the enzyme. Both the inhibition by malate, aspartate and glutamate, and the activation by sugar phosphates rises considerably when pH is decreased from 7.8 to 7.0. Malonate scarcely affects the enzyme.
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PMID:Phosphoenolpyruvate carboxylase from the roots of yellow lupin (Lupinus luteus). 667 22

The biological role of exogenous carbon dioxide during substrate assimilation with a various degree of reductivity is evaluated. The investigation of metabolic pathways of carbon dioxide incorporation into the metabolic processes of methaneoxidizing bacteria shows that the HCO3- ion assimilation is catalyzed by phosphoenolpyruvate carboxylase and in certain strains also by the key enzyme of autotrophic pathway of the carbon dioxide assimilation, ribulose-1,5-diphosphate carboxylase. The theoretical calculations and experimental studies indicate that exogenous carbon dioxide is a necessary participant of the metabolic processes of methane or methanol assimilation. It is also an acceptor of the excess electrons of these compounds. It is the degree of reductivity of the substrate metabolized that determines the activity of the exogenous carbon dioxide fixation by microorganisms. The carbon dioxide fixation by heterotrophic microorganisms must be considered, therefore, as a process which is mostly due to the elementary composition of the source of carbon under conversion.
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PMID:[Theoretical evaluation of necessity of carbon dioxide assimilation by microorganisms during growth on various substrates]. 677 May 14

A rat liver protein with both phosphoenolpyruvate carboxykinase ferroactivator activity and catalase activity has been purified to near-homogeneity. The protein has a native molecular weight of 240,000 and is composed of four identical subunits containing ferriprotoporphyrin IX prosthetic groups. The visible spectrum has absorbance maxima at 403, 500, 530, and 620 nm; it is not reduced by dithionite. The spectrum, physical properties, and specific activity are almost identical with those of catalases from other sources, and the protein has been tentatively identified as rat liver catalase. The protein exhibited partial reactivity in double immunodiffusion plates to antiserum prepared against rat liver ferroactivator isolated by a previous method (Bentle, L. A., and Lardy, H. A. (1977) J. Biol. Chem. 252, 1431-1440) raising the possibility that the original ferroactivator and rat liver catalase are structurally related. Inactivation of catalase by 3-amino-1,2,4-triazole was accompanied by loss of ferroactivator activity as well. The apparent specific activity of ferroactivator, as well. The apparent specific activity of ferroactivator, whether heme-containing or not, can be increased between 2- and 100-fold by the inclusion of bovine serum albumin, HCO3-, or a combination of the two in the incubation.
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PMID:Purification and characterization of a rat liver ferroactivator with catalase activity. 680 38

Phosphoenolpyruvate carboxykinase from bullfrog liver mitochondria has been purified to electrophoretical and immunological homogeneity by an improved method using hydrophobic chromatography on Sepharose-hexane-GMP and affinity chromatography on phosphocellulose. The molecular weight was determined to be 70,000 by SDS-gel electrophoresis, 65,000 by Sephadex G-100 gel filtration and 72,000 by glycerol gradient centrifugation. The isoelectric point was determined to be 6.2, differing from that of the cytosol enzyme. The rabbit IgG fraction against the mitochondrial PEP carboxykinase precipitated not only the mitochondrial but also the cytosol enzyme. The dissociation constant of the nucleotide-enzyme complex was determined to be 3 microM for GTP, 8.5 microM for GDP, and 171 microM for GMP. The affinity of GTP for the enzyme was reduced in the presence of phosphoenolpyruvate or Mn2+, whereas that of GDP was not changed. GMP inhibited the enzyme competitively with GDP for the phosphoenolpyruvate carboxylation and competitively with GTP for the exchange reaction between [14C]HCO3- and oxaloacetate. The purified enzyme was found to have a cysteine residue which reacted with iodoacetamide to form inactive enzyme. Guanine nucleotides or IDP and Mn2+ at a lower concentration prevented the inactivation by iodoacetamide of the enzyme in a competitive manner. Binding of guanine nucleotide to the enzyme and the relation of the sulfhydryl group to the nucleotide binding are discussed.
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PMID:Purification and molecular characteristics of mitochondrial phosphoenolpyruvate carboxykinase from bullfrog (Rana catesbeiana) liver. 697 Jan 95

Phosphoenol-3-bromopyruvate is an excellent competitive inhibitor (versus phosphoenolpyruvate) of maize phosphoenolpyruvate carboxylase (Ki = 30 microM), provided that preincubation of enzyme with inhibitor is avoided. If the enzyme is preincubated with inhibitor in the presence of Mn2+ and HCO3(-), complete inactivation occurs over the course of about 1 h. The inactivation is first order in enzyme and is saturable with respect to inhibitor, with an apparent Michaelis constant of 67.5 microM and a half-life at high inhibitor concentration of 13.4 min. The inactivation is inhibited by phospholactate and is much slower at low HCO3(-) concentration. Incubation of the enzyme with phosphoenol-3-bromopyruvate in the presence of H14CO3(-) and Mn2+ followed by treatment with NaBH4 leads to incorporation of 14C into the inactive enzyme. If treatment with NaBH4 is omitted, the enzyme is inactivated, but no 14C is incorporated into the inactive product. It appears that phosphoenol-3-bromopyruvate is a mechanism-based inactivator of phosphoenolpyruvate carboxylase, probably because of enzyme-catalyzed conversion to 3-bromooxalacetate, which alkylates the enzyme.
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PMID:Phosphoenol-3-bromopyruvate. A mechanism-based inhibitor of phosphoenolpyruvate carboxylase from maize. 717 54

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.
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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.
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PMID:Purification and characterization of phospho enol pyruvate carboxykinase from Trypanosoma brucei. 776 79

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


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