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)

Saccharomyces cerevisiae phospho enol pyruvate carboxykinase (EC 4.1.1.49), inactivated by N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine, incorporated 0.95 mol of the fluorescent moiety per mol of enzyme subunit. Reagent incorporation was completely protected by the presence of ADP plus MnCl2. The labeled protein was digested with trypsin after carboxymethylation. Two labeled peptides were isolated by reverse-phase high-performance liquid chromatography and were sequenced by gas-phase automatic Edman degradation. Both peptides contained overlapping amino acid sequences from Asn-358 to Lys-375, thus identifying Cys-364 as the reactive amino acid residue. The position of the target amino acid residue is immediately preceding a putative phosphoryl-binding sequence proposed for some nucleotide-binding proteins.
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PMID:ATP-dependent Saccharomyces cerevisiae phospho enol pyruvate carboxykinase: isolation and sequence of a peptide containing a highly reactive cysteine. 154 Jun 32

The mixed anhydride of oxalic and phosphoric acids, oxalyl phosphate, has been prepared by reaction of oxalyl chloride and inorganic phosphate in aqueous solution. The product was purified by anion exchange chromatography and characterized by 31P and 13C NMR. This acyl phosphate has a half-life of 51 h at pH 5.0 and 4 degrees C. Oxalyl phosphate, an analogue of phosphoenolpyruvate, is a slow substrate for pyruvate kinase, undergoing an enzyme-dependent phosphotransfer reaction to produce ATP from ADP. Oxalyl phosphate substitutes for phosphoenolpyruvate in the reaction catalyzed by pyruvate, phosphate dikinase. The acyl phosphate reacts with the free enzyme to give the phosphorylated form of the enzyme. Removal of the potent product inhibitor, oxalate, from the reaction mixtures by gel filtration chromatography permitted further reaction of the phosphorylated enzyme with pyrophosphate and AMP to give ATP and Pi in a single turnover assay. Oxalyl phosphate also served as a phospho group donor in a partial reaction catalyzed by phosphoenolpyruvate carboxykinase wherein GDP is phosphorylated at the expense of oxalyl phosphate.
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PMID:Synthesis of oxalyl phosphate and processing of the acyl phosphate by phosphoenolpyruvate-dependent enzymes. 216 54

The purpose of this study was to determine whether changes in ADP-ribosylation affect expression of the gene encoding the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) in H4IIE hepatoma cells. Treatment with 3-aminobenzamide, a specific inhibitor of poly(ADP ribose) synthetase, caused an 89% decrease of ADP-ribosylation in isolated nuclei, and resulted in a two- to threefold induction of immunoassayable PEPCK in cultured cells. In contrast, the structurally related compound p-aminobenzoic acid had no significant effect on either process. In vivo labeling of proteins with [35S]methionine, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, showed that the induction of immunoreactive PEPCK by 3-aminobenzamide was due to a selective increase in the synthesis of the protein. The specific induction of PEPCK synthesis by 3-aminobenzamide was accounted for by a twofold increase of mRNAPEPCK which reached its maximal value 4 h after the addition of 3-aminobenzamide and returned to the basal level by 10 h. A possible role of ADP-ribosylation in cAMP or glucocorticoid induction of PEPCK was investigated in experiments in which H4IIE cells were treated with combinations of 3-aminobenzamide and either dexamethasone or a cAMP analog. In each case the effects on PEPCK induction were additive, indicating that glucocorticoids and cAMP induce PEPCK by pathways different from that used by 3-aminobenzamide.
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PMID:3-Aminobenzamide inhibits poly(ADP ribose) synthetase activity and induces phosphoenolpyruvate carboxykinase (GTP) in H4IIE hepatoma cells. 282 39

(1) Rabbit liver mitochondria can convert exogenous phosphoenolpyruvate to malate. (2) Malate production is dependent on phosphoenolpyruvate and HCO3- and is stimulated by CN- or malonate alone and especially in combination. (3) Malate production is inhibited 70% by 3-mercaptopicolinate, a specific inhibitor of phosphoenolpyruvate carboxykinase, and 50-60% by 1,2,3-benzenetricarboxylate, an inhibitor of the tricarboxylate transporter. (4) Rat liver mitochondria incubated with phosphoenolpyruvate under identical conditions do not produce malate. (5) Malate production from phosphoenolpyruvate is stimulated by exogenous GDP or IDP but not by ADP. (6) Data support the conclusion that malate is being produced from oxalacetate generated by reversal of mitochondrial phosphoenolpyruvate carboxykinase. A possible role for this enzyme in hepatic lipogenesis is suggested.
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PMID:Synthesis of malate from phosphoenolpyruvate by rabbit liver mitochondria: implications for lipogenesis. 283 92

A cell-free system prepared from rat liver containing cytosol and mitochondria as well as a number of cofactors and gluconeogenic intermediates at near-physiological concentrations was shown to form hexose 6-phosphates linearly from lactate + pyruvate + glutamate at a rate of 0.82 +/- 0.05 mumol/min per g of liver (mean +/- S.E.M., n = 8, 37 degrees C). The indicated rates were measured between 20 min and 60 min incubation time, when the system was near steady state. Experiments with either [1-14C]lactate or [U-14C]glutamate revealed that the incorporation of radioactive label into hexose 6-phosphates was proportional to the utilization of lactate + pyruvate and of glutamate during incubation and that both served as gluconeogenic substrates at a ratio of about 2:1. When the [ATP]/[ADP] ratio was lowered from 60 to 19 by addition of ATPase, the rate of hexose 6-phosphate formation fell to one-third. This decrease in gluconeogenic flux was mainly due to a decreased flow through the phosphoglycerate kinase step. Hexose 6-phosphate formation could also be decreased by increasing the ratio [NADH]/[NAD+], either by addition of ethanol or by increasing the initial concentration of lactate + pyruvate at a fixed ratio of 10:1. The observed inhibition was linked to a limitation in the availability of oxaloacetate for the phosphoenolpyruvate carboxykinase reaction and to an increased formation of sn-glycerol 3-phosphate. Finally, the rates of hexose 6-phosphate formation in incubations with cytosols from fed rats were only 50% of those observed with cytosols from animals starved for 48 h. One of the limiting steps was found to be the flow through the phosphoenolpyruvate carboxykinase step.
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PMID:Formation of hexose 6-phosphates from lactate + pyruvate + glutamate by a cell-free system from rat liver. 287 56

Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase [ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49] is completely inactivated by the 2',3'-dialdehyde derivative of ATP (oATP) in the presence of Mn2+. The dependence of the pseudo-first-order rate constant on reagent concentration indicates the formation of a reversible complex with the enzyme (Kd = 60 +/- 17 microM) prior to covalent modification. The maximum inactivation rate constant at pH 7.5 and 30 degrees C is 0.200 +/- 0.045 min-1. ATP or ADP plus phosphoenolpyruvate effectively protect the enzyme against inactivation. oATP is a competitive inhibitor toward ADP, suggesting that oATP interacts with the enzyme at the substrate binding site. The partially inactivated enzyme shows an unaltered Km but a decreased V as compared with native phosphoenolpyruvate carboxykinase. Analysis of the inactivation rate at different H+ concentrations allowed estimation of a pKa of 8.1 for the reactive amino acid residue in the enzyme. Complete inactivation of the carboxykinase can be correlated with the incorporation of about one mole of [8-14C]oATP per mole of enzyme subunit. The results indicate that oATP can be used as an affinity label for yeast phosphoenolpyruvate carboxykinase.
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PMID:Affinity labeling of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase with the 2',3'-dialdehyde derivative of ATP. 305 40

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

Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49) is completely inactivated by phenylglyoxal and 2,3-butanedione in borate buffer at pH 8.4, with pseudo-first-order kinetics and a second-order rate constant of 144 min-1 X M-1 and 21.6 min-1 X M-1, respectively. Phosphoenolpyruvate, ADP and Mn2+ (alone or in combination) protect the enzyme against inactivation, suggesting that the modification occurs at or near to the substrate-binding site. Almost complete restoration of activity was obtained when a sample of 2,3-butanedione-inactivated enzyme was freed of excess modifier and borate ions, suggesting that only arginyl groups are modified. The changes in the rate of inactivation in the presence of substrates and Mn2+ were used to determine the dissociation constants for enzyme-ligand complexes, and values of 23 +/- 3 microM, 168 +/- 44 microM and 244 +/- 54 microM were found for the dissociation constants for the enzyme-Mn2+, enzyme-ADP and enzyme-phosphoenolpyruvate complexes, respectively. Based on kinetic data, it is shown that 1 mol of reagent must combine per enzyme active unit in order to inactivate the enzyme. Complete inactivation of the carboxykinase can be correlated with the incorporation of 3-4 mol [7-14C]phenylglyoxal per mol of enzyme subunit. Assuming a stoichiometry of 1:1 between phenylglyoxal incorporation and arginine modification, our results suggest that the modification of only two of the three to four reactive arginine residues per phosphoenolpyruvate carboxykinase subunit is responsible for inactivation.
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PMID:The presence of functional arginine residues in phosphoenolpyruvate carboxykinase from Saccharomyces cerevisiae. 330 26

The phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49) of the epimastigote form of Trypanosoma (Schizotrypanum) cruzi has been purified to homogeneity. The enzyme is composed of two apparently identical 42,000 +/- 500 subunits, is highly specific for adenine nucleotides, and has a strict requirement of Mn2+ ions for activity; the activation of the enzyme by ionic Mn2+ reveals that one Mn2+ ion required for each 42,000 subunit. Hyperbolic kinetics are observed for all substrates in the carboxylation reaction with Km (phosphoenolpyruvate) of 0.36 +/- 0.08 mM, Km (HCO-3) of 3.7 +/- 0.2 mM, and Km (Mg-ADP) of 39 +/- 1 microM. In the decarboxylation reaction the kinetics with respect to oxalacetic acid are also hyperbolic with a Km of 27 +/- 3 microM, but towards Mg-ATP there is a biphasic response: hyperbolic at low (less than 250 microM) concentrations with a Km of 39 +/- 1 microM, but at higher concentrations the nucleotide produces a strong inhibition of the enzyme activity. This inhibition is also observed with Mg-GTP and Mg-ITP which are not substrates of the reaction. The results are consistent with an important regulatory function of the enzyme in the amino-acid catabolism of T. cruzi.
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PMID:The phosphoenolpyruvate carboxykinase of Trypanosoma (Schizotrypanum) cruzi epimastigotes: molecular, kinetic, and regulatory properties. 331 Aug 97

The mechanism of C4 acid decarboxylation was studied in bundle sheath cell strands from Urochloa panicoides, a phosphoenolpyruvate carboxykinase (PCK)-type C4 plant. Added malate was decarboxylated to give pyruvate and this activity was often increased by adding ADP. Added oxaloacetate or aspartate plus 2-oxoglutarate (which produce oxaloacetate via aspartate aminotransferase) gave little metabolic decarboxylation alone but with added ATP there was a rapid production of PEP. For this activity ADP could replace ATP but only when added in combination with malate. In addition, the inclusion of aspartate plus 2-oxoglutarate with malate plus ADP often increased the rate of pyruvate production from malate by more than twofold. Experiments with respiratory chain inhibitors showed that the malate-dependent stimulation of oxaloacetate decarboxylation (PEP production) was probably due to ATP generated during the oxidation of malate in mitochondria. We could provide no evidence that photophosphorylation could serve as an alternative source of ATP for the PEP carboxykinase reaction. We concluded that both PEP carboxykinase and mitochondrial NAD-malic enzyme contribute to C4 acid decarboxylation in these cells, with the required ATP being derived from oxidation-linked phosphorylation in mitochondria.
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PMID:Photosynthesis in phosphoenolpyruvate carboxykinase-type C4 plants: pathways of C4 acid decarboxylation in bundle sheath cells of Urochloa panicoides. 334 40


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