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European Journal of Biochemistry Oct 1984Enzyme-IIIglc is part of the glucose phosphotransferase system of Escherichia coli and Salmonella typhimurium and is phosphorylated by phosphoenolpyruvate in a reaction...
Enzyme-IIIglc is part of the glucose phosphotransferase system of Escherichia coli and Salmonella typhimurium and is phosphorylated by phosphoenolpyruvate in a reaction requiring enzyme I (phosphoenolpyruvate-protein phosphotransferase), and the histidine-containing phospho-carrier protein HPr. In this paper we report the isolation of IIIglc from E. coli and the characterization of the active center. Alkaline hydrolysis of [32P]P-IIIglc and chromatography of the hydrolysate suggested that the phosphoryl group is bound to a histidyl residue in P-IIIglc of S. typhimurium. Here we present 1H-NMR measurements of IIIglc and P-IIIglc from E. coli which further substantiate that the phosphoryl group in P-IIIglc is linked to the N-3 position of a histidyl residue. After phosphorylation of IIIglc with [32P]Phosphoenolpyruvate, enzyme I and HPr, the phosphorylated protein was cleaved with either alkaline protease from Streptomyces griseus or subtilisin from Bacillus subtilis. According to amino acid analysis both proteases produced the same peptide carrying the phosphoryl group. The amino acid sequence of this peptide was found to be Val-His-Phe-Gly-Ile-Asp. The lower electrophoretic mobility of P-IIIglc on dodecylsulfate/polyacrylamide gels and its stronger binding to the hydrophobic matrix of a reversed-phase column compared to unphosphorylated protein may indicate a structural change following phosphoenolpyruvate-dependent phosphorylation.
Topics: Binding Sites; Chemical Phenomena; Chemistry; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Escherichia coli Proteins; Magnetic Resonance Spectroscopy; Peptides; Phosphoenolpyruvate; Phosphoenolpyruvate Sugar Phosphotransferase System; Phosphorylation
PubMed: 6383826
DOI: 10.1111/j.1432-1033.1984.tb08438.x -
Biochimica Et Biophysica Acta Apr 1964
Topics: Carbohydrate Metabolism; Glycerophosphates; Metabolism; Muscle Contraction; Muscles; Phosphates; Phosphoenolpyruvate; Pyruvates
PubMed: 14166875
DOI: 10.1016/0304-4165(64)90161-8 -
Biochemistry Feb 1988(Z)-3-(Fluoromethyl)phosphoenolpyruvate has been synthesized in nine chemical steps from glyoxylic acid. The compound is stable at pH 3, but at pH 8 it decomposes within...
(Z)-3-(Fluoromethyl)phosphoenolpyruvate has been synthesized in nine chemical steps from glyoxylic acid. The compound is stable at pH 3, but at pH 8 it decomposes within seconds to give 2-oxo-3-butenoate. When 3-(fluoromethyl)phosphoenolpyruvate is added to a solution of phosphoenolpyruvate carboxylase or pyruvate kinase, the enzyme is inactivated over the course of an hour. Identical kinetics of inactivation are observed whether the reaction is initiated by addition of 3-(fluoromethyl)-phosphoenolpyruvate, preformed 2-oxo-3-butenoate, or 4-fluoro-2-oxobutanoate (which rapidly undergoes elimination of fluoride ion to form 2-oxo-3-butenoate). The inactivating species in all cases is believed to be 2-oxo-3-butenoate. The inactivation is completely prevented by the presence of dithiothreitol, which reacts rapidly with 2-oxo-3-butenoate. Studies with competitive inhibitors of both enzymes indicate that inactivation does not occur at the active site.
Topics: Carboxy-Lyases; Indicators and Reagents; Kinetics; L-Lactate Dehydrogenase; Magnetic Resonance Spectroscopy; Phosphoenolpyruvate; Phosphoenolpyruvate Carboxylase; Pyruvate Kinase; Structure-Activity Relationship; Substrate Specificity
PubMed: 3365390
DOI: 10.1021/bi00404a039 -
Seikagaku. the Journal of Japanese... Jun 1984
Review
Topics: 4-Chloromercuribenzenesulfonate; Biological Transport, Active; Cell Membrane Permeability; Erythrocyte Membrane; Humans; Hydrogen-Ion Concentration; Phosphoenolpyruvate; Spherocytosis, Hereditary; Temperature
PubMed: 6090549
DOI: No ID Found -
Cell Reports Methods May 2024Co-assembling enzymes with nanoparticles (NPs) into nanoclusters allows them to access channeling, a highly efficient form of multienzyme catalysis. Using pyruvate...
Co-assembling enzymes with nanoparticles (NPs) into nanoclusters allows them to access channeling, a highly efficient form of multienzyme catalysis. Using pyruvate kinase (PykA) and lactate dehydrogenase (LDH) to convert phosphoenolpyruvic acid to lactic acid with semiconductor quantum dots (QDs) confirms how enzyme cluster formation dictates the rate of coupled catalytic flux (k) across a series of differentially sized/shaped QDs and 2D nanoplatelets (NPLs). Enzyme kinetics and coupled flux were used to demonstrate that by mixing different NP systems into clusters, a >10× improvement in k is observed relative to free enzymes, which is also ≥2× greater than enhancement on individual NPs. Cluster formation was characterized with gel electrophoresis and transmission electron microscopy (TEM) imaging. The generalizability of this mixed-NP approach to improving flux is confirmed by application to a seven-enzyme system. This represents a powerful approach for accessing channeling with almost any choice of enzymes constituting a multienzyme cascade.
Topics: L-Lactate Dehydrogenase; Lactic Acid; Pyruvate Kinase; Nanoparticles; Phosphoenolpyruvate; Quantum Dots; Kinetics
PubMed: 38714198
DOI: 10.1016/j.crmeth.2024.100764 -
Journal of Molecular Graphics &... Dec 2020The shikimate pathway consists of seven enzymatic steps involved in the conversion of erythrose-4-phosphate and phosphoenolpyruvate to chorismate and also responsible to...
The shikimate pathway consists of seven enzymatic steps involved in the conversion of erythrose-4-phosphate and phosphoenolpyruvate to chorismate and also responsible to the production of aromatic amino acids, such as phenylalanine, tyrosine, and tryptophan which are essential to the bacterial metabolism. The 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS) and 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) catalyze important steps in the shikimate pathway using as substrate the phosphoenolpyruvate (PEP). Due to the importance of PEP in shikimate pathway, its structure has been investigated to develop new bioinspired competitive inhibitors against DAHPS and EPSPS. In the present study, we perform a literature survey of 28 PEP derivatives, then we analyzed the selectivity and affinity of these compounds against the EPSPS and DAHPS structures using consensual molecular docking, pharmacophore prediction, molecular dynamics (MD) simulations, and binding free energy calculations. Here, we propose consistent binding modes of the selected ligands and indicate that their structures show interesting pharmacophoric properties related to multi-targets inhibitors for both enzymes. Our computational results are supported by previous experimental findings related to the interactions of PEP derivatives with DAHPS and EPSPS structures.
Topics: 3-Deoxy-7-Phosphoheptulonate Synthase; 3-Phosphoshikimate 1-Carboxyvinyltransferase; Molecular Docking Simulation; Phosphoenolpyruvate; Shikimic Acid
PubMed: 32947107
DOI: 10.1016/j.jmgm.2020.107735 -
Biochemistry Feb 1983Phosphoenolpyruvate when heated in acidic solution exchanges its phosphoryl and carboxyl oxygens rapidly and its enolic oxygen much more slowly with oxygens from water.... (Comparative Study)
Comparative Study
Phosphoenolpyruvate when heated in acidic solution exchanges its phosphoryl and carboxyl oxygens rapidly and its enolic oxygen much more slowly with oxygens from water. The incorporation of 18O into phosphoenolpyruvate was measured by gas chromatography-mass spectrometry and phosphorus-31 nuclear magnetic resonance after heating in H218O at 98 degrees C. The rates of exchange of all six oxygens of phosphoenolpyruvate with water increase with increasing acidity, and the phosphoryl oxygens exchange more rapidly than the carboxyl oxygens. The rate of exchange of each oxygen of the phosphoryl group is 16-fold greater than the hydrolysis rate at 1 N HCl. This provides a simple and useful method for the synthesis of [18O]phosphoenolpyruvate highly enriched in its phosphoryl-group oxygens. An enrichment of 89% was obtained with a 50% yield. The [18O]-phosphoenolpyruvate showed a binomial distribution of 18O in the phosphoryl-group oxygens. The exchange may be explained by the reversible formation of a transient cyclic phosphate and, for exchange of the enolic oxygen, a transient acyl phosphate. Preparation of [18O]phosphoenolypyruvate from [18O]Pi by a chemical synthesis from beta-chlorolactate was not satisfactory because of drastic loss of 18O during the procedures used. Some loss of 18O also occurred during an enzymic synthesis with KCNO, [18O]Pi, carbamate kinase, and pyruvate kinase.
Topics: Gas Chromatography-Mass Spectrometry; Hydrolysis; In Vitro Techniques; Magnetic Resonance Spectroscopy; Oxygen Isotopes; Phosphoenolpyruvate
PubMed: 6838816
DOI: 10.1021/bi00272a013 -
Plant Physiology Sep 2011Day respiration is the cornerstone of nitrogen assimilation since it provides carbon skeletons to primary metabolism for glutamate (Glu) and glutamine synthesis....
Day respiration is the cornerstone of nitrogen assimilation since it provides carbon skeletons to primary metabolism for glutamate (Glu) and glutamine synthesis. However, recent studies have suggested that the tricarboxylic acid pathway is rate limiting and mitochondrial pyruvate dehydrogenation is partly inhibited in the light. Pyruvate may serve as a carbon source for amino acid (e.g. alanine) or fatty acid synthesis, but pyruvate metabolism is not well documented, and neither is the possible resynthesis of phosphoenolpyruvate (PEP). Here, we examined the capacity of pyruvate to convert back to PEP using (13)C and (2)H labeling in illuminated cocklebur (Xanthium strumarium) leaves. We show that the intramolecular labeling pattern in Glu, 2-oxoglutarate, and malate after (13)C-3-pyruvate feeding was consistent with (13)C redistribution from PEP via the PEP-carboxylase reaction. Furthermore, the deuterium loss in Glu after (2)H(3)-(13)C-3-pyruvate feeding suggests that conversion to PEP and back to pyruvate washed out (2)H atoms to the solvent. Our results demonstrate that in cocklebur leaves, PEP resynthesis occurred as a flux from pyruvate, approximately 0.5‰ of the net CO(2) assimilation rate. This is likely to involve pyruvate inorganic phosphate dikinase and the fundamental importance of this flux for PEP and inorganic phosphate homeostasis is discussed.
Topics: Carbon Isotopes; Phosphoenolpyruvate; Plant Leaves; Pyruvic Acid
PubMed: 21730197
DOI: 10.1104/pp.111.180711 -
Acta Crystallographica. Section D,... Dec 2015Staphylococcus aureus is a Gram-positive bacterium with strong pathogenicity that causes a wide range of infections and diseases. Enolase is an evolutionarily conserved...
Staphylococcus aureus is a Gram-positive bacterium with strong pathogenicity that causes a wide range of infections and diseases. Enolase is an evolutionarily conserved enzyme that plays a key role in energy production through glycolysis. Additionally, enolase is located on the surface of S. aureus and is involved in processes leading to infection. Here, crystal structures of Sa_enolase with and without bound phosphoenolpyruvate (PEP) are presented at 1.6 and 2.45 Å resolution, respectively. The structure reveals an octameric arrangement; however, both dimeric and octameric conformations were observed in solution. Furthermore, enzyme-activity assays show that only the octameric variant is catalytically active. Biochemical and structural studies indicate that the octameric form of Sa_enolase is enzymatically active in vitro and likely also in vivo, while the dimeric form is catalytically inactive and may be involved in other biological processes.
Topics: Amino Acid Motifs; Bacterial Proteins; Catalytic Domain; Crystallography, X-Ray; Escherichia coli; Gene Expression; Kinetics; Models, Molecular; Molecular Sequence Data; Phosphoenolpyruvate; Phosphopyruvate Hydratase; Protein Binding; Protein Multimerization; Protein Structure, Secondary; Recombinant Proteins; Staphylococcus aureus
PubMed: 26627653
DOI: 10.1107/S1399004715018830 -
Acta Crystallographica. Section C,... Jul 2000
Topics: Crystallography, X-Ray; Models, Molecular; Molecular Conformation; Phosphoenolpyruvate; Water
PubMed: 10935091
DOI: 10.1107/s010827010000620x