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Biochemistry Apr 1996Phosphoenolpyruvate phosphomutase (PEP mutase) from Tetrahymena pyriformis catalyzes the rearrangement of phosphoenolpyruvate (PEP) to phosphonopyruvate (P-pyr). A...
Phosphoenolpyruvate phosphomutase (PEP mutase) from Tetrahymena pyriformis catalyzes the rearrangement of phosphoenolpyruvate (PEP) to phosphonopyruvate (P-pyr). A spectrophotometric P-pyr assay consisting of the coupled actions of P-pyr decarboxylase, phosphonoacetaldehyde hydrolase, and alcohol dehydrogenase was devised to monitor mutase catalysis. The reaction constants determined for PEP mutase catalyzed conversion of PEP to P-pyr at pH 7.5 and 25 degrees C in the presence of Mg(II) are kcat = 5 s(-1), Km = 0.77 +/- 0.05 mM, and Keq = (2-9) x 10(-4). In the PEP forming direction, kcat = 100 s(-1) and Km = 3.5 +/- 0.1 microM. Retention of stereochemistry at phosphorus and strong inhibition displayed by the pyruvyl enolate analog, oxalate, have been cited as two lines of evidence that PEP mutase catalysis proceeds via a phosphoenzyme-pyruvyl enolate intermediate [Seidel, H. M., & Knowles, J. R. (1994) Biochemistry 33, 5641-5646]. In this study, single turnover reactions of oxalyl phosphate with the PEP mutase were carried out to test the formation of the phosphoenzyme intermediate. If formed. the phosphoenzyme-oxalate complex should be sufficiently stable to isolate. Reaction of the mutase with [32P]oxalyl phosphate in the presence of Mg(II)/Mn(II) cofactor failed to produce a detectable level of the [32P]phosphoenzyme-oxalate complex. In contrast, the same reaction carried out with pyruvate phosphate dikinase (PPDK), an enzyme known to catalyze the phosphorylation of its active site histidine with PEP, occurred at a rate of 4 x 10(-4) s(-1) (15% E-P formed) in the presence Mg(II) and at a rate of 3 x 10(-3) s(-1) (60% E-P formed) in the presence of Mn(II). Both oxalyl phosphate (Ki = 180 +/- 10 microM) and oxalate (Ki = 32 +/- 1O microM) were competitive inhibitors of PEP mutase catalysis, but neither displayed slow, tight binding inhibition. These results do not support the intermediacy of a phosphoenzyme-pyruvyl enolate complex in PEP mutase catalysis.
Topics: Animals; Aspartate Aminotransferases; Binding Sites; Carbon; Catalysis; Energy Metabolism; Kinetics; Magnetic Resonance Spectroscopy; Malate Dehydrogenase; Molecular Structure; Phosphoenolpyruvate; Phosphorus; Phosphotransferases (Phosphomutases); Spectrophotometry; Tetrahymena pyriformis; Thermodynamics
PubMed: 8605214
DOI: 10.1021/bi952944k -
Journal of Enzyme Inhibition and... Dec 2016A bioautographic assay based on thin layer chromatography was developed for phosphoenolpyruvate (PEP) detecting as a known but rarely studied inhibitor of phosphoglucose...
A bioautographic assay based on thin layer chromatography was developed for phosphoenolpyruvate (PEP) detecting as a known but rarely studied inhibitor of phosphoglucose isomerase (PGI). The protocol with NADP(+)/NBT/PMS (β-nicotinamide adenine dinucleotide phosphate/nitrotetrazolium blue chloride/phenazine methosulfate) staining was capable of detecting Mycobacterium tuberculosis H37Ra PGI inhibition using PEP. According to this method, visibly brighter spots (zones) against purple background are observed in the area of inhibition of the above-mentioned enzyme activity. The detection limit for PEP as an inhibitor of Mycobacterium tuberculosis H37Ra PGI was 226 μg per spot/zone. Noteworthy is that we are the first authors to have successfully used a bioautographic assay to detect Mycobacterium tuberculosis H37Ra PGI inhibition by PEP.
Topics: Chromatography, Thin Layer; Enzyme Inhibitors; Glucose-6-Phosphate Isomerase; Mycobacterium tuberculosis; Phosphoenolpyruvate
PubMed: 26986942
DOI: 10.3109/14756366.2016.1151012 -
Proceedings of the National Academy of... Nov 1994Nonmetabolizable glucose analogs inhibit the growth of Escherichia coli on a wide variety of carbon sources. This phenomenon was investigated with particular reference...
Nonmetabolizable glucose analogs inhibit the growth of Escherichia coli on a wide variety of carbon sources. This phenomenon was investigated with particular reference to the effect of 2-deoxyglucose (2DG) on growth on fructose as sole carbon source. When the inhibitor is supplied in sufficiently low concentrations, the initial arrest of growth is overcome; this relief of inhibition is aided by means that increase the availability of phosphoenolpyruvate (PEP) to the growing cells, such as the use of L-aspartate instead of ammonium chloride as sole nitrogen source for growth, and the introduction of the pps+ allele into a pps- strain. Studies with [14C]2DG showed that the analog or its 6-phosphate as such did not inhibit growth but that 2DG exerted its effect by competing for intracellular PEP and lowering its concentration below that needed to sustain growth. Direct measurements of the PEP-dependent phosphorylation of 2DG and of fructose by permeabilized E. coli showed that the apparent Km for PEP was nearly 7 times higher for 2DG that it was for fructose, although the apparent Vmax for 2DG was nearly 3 times that for fructose; this explains the ability of cells to overcome the inhibition by low, but not by high, concentrations of 2DG.
Topics: Aspartic Acid; Biological Transport; Deoxyglucose; Escherichia coli; Fructose; Kinetics; Phosphoenolpyruvate; Phosphoenolpyruvate Sugar Phosphotransferase System; Phosphotransferases (Paired Acceptors)
PubMed: 7972013
DOI: 10.1073/pnas.91.23.11080 -
FEBS Letters Oct 1983Phosphoenolpyruvate-dependent protein kinase activity has been demonstrated in the soluble fraction of rat skeletal muscle. The reaction was not due to the formation of...
Phosphoenolpyruvate-dependent protein kinase activity has been demonstrated in the soluble fraction of rat skeletal muscle. The reaction was not due to the formation of ATP in the incubation mixture. Cyclic AMP, calcium, ATP and a number of phosphate acceptor proteins did not stimulate the reaction. One 32P-labelled protein (Mr 25000) was observed on SDS gels. The phosphorylated protein contained acid stable phosphoserine as a major phosphorylated amino acid. The phosphorylation reaction in crude extracts was not directly proportional to the amount of protein, but typical of a two-component system; i.e., kinase and substrate. The chromatography of soluble proteins on Ultrogel AcA44 separated the phosphate acceptor protein(s) from the phosphoenolpyruvate-dependent protein kinase activity.
Topics: Adenosine Triphosphate; Animals; Muscle Proteins; Muscles; Phosphoenolpyruvate; Phosphorylation; Phosphoserine; Protein Kinases; Rats
PubMed: 6617885
DOI: 10.1016/0014-5793(83)81063-1 -
Acta Crystallographica. Section B,... Oct 1996The crystal structure of (C6H11NH3+)3. Pep3-.H2O, where Pep3- = (O-)2P(O)-O-C(CH2)-CO2-, is reported and the systematic structural variations among 19 crystallographic...
The crystal structure of (C6H11NH3+)3. Pep3-.H2O, where Pep3- = (O-)2P(O)-O-C(CH2)-CO2-, is reported and the systematic structural variations among 19 crystallographic occurrences of H3Pep, H2Pep-, HPep2- and Pep3- species, which are important phosphate donors in the ATP cycle of bioenergetics, are reviewed. Tris(cyclohexylammonium) phosphoenolpyruvate monohydrate, (C6H11NH3+)3.-[O3POC(CH2)CO2]3-.H2O, M(r) = 483.6, m.p. 418-420K; T = 296(1)K; orthorhombic, P2(1)2(1)2(1); a = 16.7042(5), b = 24.4881 (6), c = 6.38910 (10) A; V = 2613.49(11) A3, Z = 4, Dx = 1.23, Dm = 1.22 mg mm-3, mu = 0.14 mm-1 for lambda(MoK alpha) = 0.7107 A; F(000) = 1056 e; R(magnitude of F) = 0.0608 for 6056 hkl and hkl data with (sin theta)/lambda < or = 0.65 A-1.
Topics: Crystallography, X-Ray; Hydrogen Bonding; Molecular Structure; Phosphoenolpyruvate
PubMed: 8900032
DOI: 10.1107/s0108768196005241 -
Journal of Chromatography Dec 1989A rapid and efficient isocratic high-performance liquid chromatographic method for studying the metabolism of phosphoenolpyruvate and uridine diphosphate glucose (UDPG)...
Isocratic high-performance liquid chromatographic determination of the concentration and specific radioactivity of phosphoenolpyruvate and uridine diphosphate glucose in tissue extracts.
A rapid and efficient isocratic high-performance liquid chromatographic method for studying the metabolism of phosphoenolpyruvate and uridine diphosphate glucose (UDPG) has been developed. For each compound this method can measure tissue concentrations in the range 0.1-1000 nmol/g of tissue and determine specific radioactivity. All measurements can be performed in 200 mg of tissue. The recoveries of uridine diphosphate [6-3H]glucose and phosphoenol[1-14C]pyruvate from liver tissue homogenates were 97 and 99%, respectively. Following intra-arterial infusion of [6-3H]glucose and [U-14C]lactate in conscious rat, the concentration and specific radioactivity of phosphoenolpyruvate and UDPG were determined in rat liver. The method may be applied to experimentation in small animals using radiolabelled precursors in order to quantitate in vivo the glycogenic and gluconeogenic fluxes.
Topics: Animals; Chromatography, High Pressure Liquid; Male; Phosphoenolpyruvate; Radiometry; Rats; Rats, Inbred Strains; Uridine Diphosphate Sugars
PubMed: 2625480
DOI: 10.1016/0378-4347(89)80006-4 -
The Biochemical Journal Sep 19781. The properties of pyruvate kinase and, if present, phosphoenolpyruvate carboxykinase from the muscles of the sea anemone, scallop, oyster, crab, lobster and frog were... (Comparative Study)
Comparative Study
Properties of pyruvate kinase and phosphoenolpyruvate carboxykinase in relation to the direction and regulation of phosphoenolpyruvate metabolism in muscles of the frog and marine invertebrates.
1. The properties of pyruvate kinase and, if present, phosphoenolpyruvate carboxykinase from the muscles of the sea anemone, scallop, oyster, crab, lobster and frog were investigated. 2. In general, the properties of pyruvate kinase from all muscles were similar, except for those of the enzyme from the oyster (adductor muscle); the pH optima were between 7.1 and 7.4, whereas that for oyster was 8.2; fructose bisphosphate lowered the optimum pH of the oyster enzyme from 8.2 to 7.1, but it had no effect on the enzymes from other muscles. Hill coefficients for the effect of the concentration of phosphoenolpyruvate were close to unity in the absence of added alanine for the enzymes from all muscles except oyster adductor muscle; it was 1.5 for this enzyme. Alanine inhibited the enzyme from all muscles except the frog; this inhibition was relieved by fructose bisphosphate. Low concentrations of alanine were very effective with the enzyme from the oyster (50% inhibition was observed at 0.4mm). Fructose bisphosphate activated the enzyme from all muscles, but extremely low concentrations were effective with the oyster enzyme (0.13mum produced 50% activation). 3. In general, the properties of phosphoenolpyruvate carboxykinase from the sea anemone and oyster muscles are similar: the K(m) values for phosphoenolpyruvate are low (0.10 and 0.13mm); the enzymes require Mn(2+) in addition to Mg(2+) for activity; and ITP inhibits the enzymes and the inhibition is relieved by alanine. These latter compounds had no effect on enzymes from other muscles. 4. It is suggested that changes in concentrations of fructose bisphosphate, alanine and ITP produce a coordinated mechanism of control of the activities of pyruvate kinase and phosphoenolpyruvate carboxykinase in the sea anemone and oyster muscles, which ensures that phosphoenolpyruvate is converted into oxaloacetate and then into succinate in these muscles under anaerobic conditions. 5. It is suggested that in the muscles of the crab, lobster and frog, phosphoenolpyruvate carboxykinase catalyses the conversion of oxaloacetate into phosphoenolpyruvate. This may be part of a pathway for the oxidation of some amino acids in these muscles.
Topics: Alanine; Animals; Anura; Cations; Hexosephosphates; Hydrogen-Ion Concentration; In Vitro Techniques; Inosine Triphosphate; Invertebrates; Kinetics; Muscles; Phosphoenolpyruvate; Phosphoenolpyruvate Carboxykinase (GTP); Pyruvate Kinase; Vertebrates
PubMed: 31870
DOI: 10.1042/bj1740979 -
Talanta Aug 2024Phosphoenolpyruvate (PEP) is an essential intermediate metabolite that is involved in various vital biochemical reactions. However, achieving the direct and accurate...
Highly sensitive and accurate measurement of underivatized phosphoenolpyruvate in plasma and serum via EDTA-facilitated hydrophilic interaction liquid chromatography-tandem mass spectrometry.
Phosphoenolpyruvate (PEP) is an essential intermediate metabolite that is involved in various vital biochemical reactions. However, achieving the direct and accurate quantification of PEP in plasma or serum poses a significant challenge owing to its strong polarity and metal affinity. In this study, a sensitive method for the direct determination of PEP in plasma and serum based on ethylenediaminetetraacetic acid (EDTA)-facilitated hydrophilic interaction liquid chromatography-tandem mass spectrometry was developed. Superior chromatographic retention and peak shapes were achieved using a zwitterionic stationary-phase HILIC column with a metal-inert inner surface. Efficient dechelation of PEP-metal complexes in serum/plasma samples was achieved through the introduction of EDTA, resulting in a significant enhancement of the PEP signal. A PEP isotopically labelled standard was employed as a surrogate analyte for the determination of endogenous PEP, and validation assessments proved the sensitivity, selectivity, and reproducibility of this method. The method was applied to the comparative quantification of PEP in plasma and serum samples from mice and rats, as well as in HepG2 cells, HEK293T cells, and erythrocytes; the results confirmed its applicability in PEP-related biomedical research. The developed method can quantify PEP in diverse biological matrices, providing a feasible opportunity to investigate the role of PEP in relevant biomedical research.
Topics: Tandem Mass Spectrometry; Animals; Humans; Hydrophobic and Hydrophilic Interactions; Edetic Acid; Mice; Chromatography, Liquid; Rats; Phosphoenolpyruvate; HEK293 Cells; Hep G2 Cells; Rats, Sprague-Dawley; Male
PubMed: 38692044
DOI: 10.1016/j.talanta.2024.126134 -
Biochemistry Jul 1992The catalytic mechanism of phosphoenolpyruvate (PEP) carboxylase from Zea mays has been studied using (Z)- and (E)-3-fluorophosphoenolpyruvate (F-PEP) as substrates....
The catalytic mechanism of phosphoenolpyruvate (PEP) carboxylase from Zea mays has been studied using (Z)- and (E)-3-fluorophosphoenolpyruvate (F-PEP) as substrates. Both (Z)- and (E)-F-PEP partition between carboxylation to produce 3-fluorooxalacetate and hydrolysis to produce 3-fluoropyruvate. Carboxylation accounts for 3% of the reaction observed with (Z)-F-PEP, resulting in the formation of (R)-3-fluorooxalacetate, and for 86% of the reaction of (E)-F-PEP forming (S)-3-fluorooxalacetate. Carboxylation of F-PEP occurs on the 2-re face, which corresponds to the 2-si face of PEP. The partitioning of F-PEP between carboxylation and hydrolysis is insensitive to pH but varies with metal ion. Use of 18O-labeled bicarbonate produces phosphate that is multiply labeled with 18O; in addition, 18O is also incorporated into residual (Z)- and (E)-F-PEP. The 13(V/K) isotope effect on the carboxylation of F-PEP catalyzed by PEP carboxylase at pH 8.0, 25 degrees C, is 1.049 +/- 0.003 for (Z)-F-PEP and 1.009 +/- 0.006 for (E)-F-PEP. These results are consistent with a mechanism in which carboxylation of PEP occurs via attack of the enolate of pyruvate on CO2 rather than carboxy phosphate. In this mechanism phosphorylation of bicarbonate to give carboxy phosphate and decarboxylation of the latter are reversible steps. An irreversible step, however, precedes partitioning between carboxylation to give oxalacetate and release of CO2, which results in hydrolysis of PEP.
Topics: Bicarbonates; Carbon Dioxide; Cations, Divalent; Hydrogen-Ion Concentration; Hydrolysis; Isomerism; Kinetics; Magnetic Resonance Spectroscopy; Malate Dehydrogenase; Phosphoenolpyruvate; Phosphoenolpyruvate Carboxylase; Structure-Activity Relationship; Zea mays
PubMed: 1633157
DOI: 10.1021/bi00143a011 -
Biochemistry Oct 1982The overall stereochemical course of the reactions leading to the phosphorylation of methyl alpha-D-glucopyranoside by the glucose-specific enzyme II (enzyme IIGlc) of...
The overall stereochemical course of the reactions leading to the phosphorylation of methyl alpha-D-glucopyranoside by the glucose-specific enzyme II (enzyme IIGlc) of the Escherichia coli phosphotransferase system has been investigated. With [(R)-16O,17O,18O]phosphoenolpyruvate as the phosphoryl donor and in the presence of enzyme I, HPr, and enzyme IIIGlc of the phosphotransferase system, membranes from E. coli containing enzyme IIGlc catalyzed the formation of methyl alpha-D-glucopyranoside 6-phosphate with overall inversion of the configuration at phosphorus (with respect to phosphoenolpyruvate). It has previously been shown that sequential covalent transfer of the phosphoryl group of phosphoenolpyruvate to enzyme I, to HPr, and to enzyme IIIGlc occurs before the final transfer from phospho-enzyme IIIGlc to the sugar, catalyzed by enzyme IIGlc. Because overall inversion of the configuration of the chiral phospho group of phosphoenolpyruvate implies an odd number of transfer steps, the phospho group has been transferred at least five times, and transfer from phospho-enzyme IIIGlc to the sugar must occur in two steps (or a multiple thereof). On the basis that no membrane protein other than enzyme IIGlc is directly involved in the final phospho transfer steps, our results imply that a covalent phospho-enzyme IIGlc is an intermediate during transport and phosphorylation of glucose by the E. coli phosphotransferase system.
Topics: Biological Transport; Cell Membrane; Escherichia coli; Escherichia coli Proteins; Glucose; Magnetic Resonance Spectroscopy; Phosphoenolpyruvate; Phosphoenolpyruvate Sugar Phosphotransferase System; Phosphorylation; Phosphotransferases (Nitrogenous Group Acceptor); Stereoisomerism
PubMed: 6756472
DOI: 10.1021/bi00265a026