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Journal of Biochemistry Jul 1997Phosphoenolpyruvate carboxylase (PEPC) was purified from an extremely thermophilic bacterium, Rhodothermus obamensis, growing optimally at 80 degrees C, which had...
Phosphoenolpyruvate carboxylase (PEPC) was purified from an extremely thermophilic bacterium, Rhodothermus obamensis, growing optimally at 80 degrees C, which had recently been isolated from a shallow marine hydrothermal vent in Japan. The native enzyme was a homotetramer of 400 kDa in molecular mass, as estimated by gel filtration chromatography, and the subunit exhibited an apparent molecular mass of 100 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum temperature for enzyme activity was 75 degrees C. The enzyme exhibited an absolute requirement for divalent cations and a pH optimum of 8.0. The enzyme was extremely thermostable and there was no loss of enzyme activity on incubation for 2 h at 85 degrees C. The enzyme exhibited a positive allosteric property with acetyl-CoA and fructose 1,6-bisphosphate, and a negative one with L-aspartate and L-malate. These effectors affected not only the thermophilicity but also the thermostability of the enzyme, and the substrate, co-factors, and salts increased the thermostability as well. The extrinsic thermostabilization might be a possible mechanism for adaptation of the enzyme to high temperature.
Topics: Acetyl Coenzyme A; Aspartic Acid; Cations; Dimerization; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Gram-Negative Aerobic Bacteria; Hydrogen-Ion Concentration; Molecular Weight; Phosphoenolpyruvate; Phosphoenolpyruvate Carboxylase; Temperature
PubMed: 9276668
DOI: 10.1093/oxfordjournals.jbchem.a021737 -
Journal of Bacteriology Feb 2007Phosphoenolpyruvate inhibited Escherichia coli NADP-isocitrate dehydrogenase allosterically (Ki of 0.31 mM) and isocitrate lyase uncompetitively (Ki' of 0.893 mM)....
Phosphoenolpyruvate inhibited Escherichia coli NADP-isocitrate dehydrogenase allosterically (Ki of 0.31 mM) and isocitrate lyase uncompetitively (Ki' of 0.893 mM). Phosphoenolpyruvate enhances the uncompetitive inhibition of isocitrate lyase by increasing isocitrate, which protects isocitrate dehydrogenase from the inhibition, and contributes to the control through the tricarboxylic acid cycle and glyoxylate shunt.
Topics: Escherichia coli; Isocitrate Dehydrogenase; Isocitrate Lyase; Isocitrates; Kinetics; NADP; Phosphoenolpyruvate
PubMed: 17142397
DOI: 10.1128/JB.01628-06 -
Chemotherapy 1977Fosfomycin, a nontoxic broad-spectrum antibiotic, different in structure from all previously described antibiotics, acts selectively by inhibiting cell wall formation....
Fosfomycin, a nontoxic broad-spectrum antibiotic, different in structure from all previously described antibiotics, acts selectively by inhibiting cell wall formation. It was overlooked during many years of screening because of antagonism by culture medium ingredients and frequent occurrence of resistant mutants. It is effective in many because the neutralizing substances are not present and resistant mutants of most species are avirulent. Fosfomycin has favorable pharmacologic characteristics. It is not cross resistant, does not show antagonism, and has been used successfully in combinations. An insoluble calcium salt is used in oral formulation and a sodium salt for parenteral administration. Overall success rates of 86% were reported with 1,000 patients in Spain and 79% in Japan.
Topics: Animals; Anti-Bacterial Agents; Bacteria; Bacterial Infections; Cell Wall; Drug Therapy, Combination; Fosfomycin; Humans; Penicillin Resistance; Penicillins; Phosphoenolpyruvate
PubMed: 583866
DOI: 10.1159/000222020 -
Analytical Biochemistry Mar 1975
Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Charcoal; Firefly Luciferin; Luciferases; Male; Mitochondria, Liver; Phosphoenolpyruvate; Pyruvate Kinase; Rats; Tricarboxylic Acids
PubMed: 1137082
DOI: 10.1016/0003-2697(75)90412-1 -
Biochemistry Feb 1985Enzyme I, the phosphoenolpyruvate:protein phosphotransferase (EC 2.7.3.9), which is part of the bacterial phosphoenolpyruvate- (PEP) dependent phosphotransferase system,...
Enzyme I, the phosphoenolpyruvate:protein phosphotransferase (EC 2.7.3.9), which is part of the bacterial phosphoenolpyruvate- (PEP) dependent phosphotransferase system, has been purified from Streptococcus faecalis by using a large-scale preparation. Size exclusion chromatography revealed a molecular weight of 140 000. On sodium dodecyl sulfate gels, enzyme I gave one band with a molecular weight of 70 000, indicating that enzyme I consists of two identical subunits. The first 59 amino acids of the amino-terminal part of the protein have been sequenced. It showed some similarities with enzyme I of Salmonella typhimurium. The active center of enzyme I has also been determined. After phosphorylation with [32P]PEP, the enzyme was cleaved by using different proteases. Labeled peptides were isolated by high-performance liquid chromatography on a reversed-phase column. The amino acid composition or amino acid sequence of the peptides has been determined. The largest labeled peptide was obtained with Lys-C protease and had the following sequence: -Ala-Phe-Val-Thr-Asp-Ile-Gly- Gly-Arg-Thr-Ser-His*-Ser-Ala-Ile-Met-Ala-Arg-Ser-Leu-Glu-Ile-Pro-Ala- Ile-Val-Gly-Thr-Lys-. It has previously been shown that the phosphoryl group is bound to the N-3 position of a histidyl residue in phosphorylated enzyme I. The single His in position 12 of the above peptide must therefore carry the phosphoryl group.
Topics: Amino Acid Sequence; Binding Sites; Chromatography, Paper; Enterococcus faecalis; Macromolecular Substances; Molecular Weight; Peptide Fragments; Phosphoenolpyruvate; Phosphoenolpyruvate Sugar Phosphotransferase System; Phosphorus Radioisotopes; Phosphotransferases (Nitrogenous Group Acceptor)
PubMed: 3922407
DOI: 10.1021/bi00325a023 -
Journal of Biochemistry Mar 1984An investigation was performed to elucidate some unusual phenomena which had been observed with phosphoenolpyruvate (PEP) carboxylase [EC 4.1.1.31] of Escherichia coli....
Phosphoenolpyruvate carboxylase of Escherichia coli. Specificity of some compounds as activators at the site for fructose 1,6-bisphosphate, one of the allosteric effectors.
An investigation was performed to elucidate some unusual phenomena which had been observed with phosphoenolpyruvate (PEP) carboxylase [EC 4.1.1.31] of Escherichia coli. (i) Fructose 1,6-bisphosphate (Fru-1,6-P2) and GTP--the allosteric activators--were competitive with each other in the activation. (ii) Some analogs of PEP such as DL-2-phospholactate and 2-phosphoglycolate, which behaved as inhibitors in the presence of the activator (acetyl-CoA or dioxane), activated the enzyme to some extent in the absence of the activator. (iii) Ammonium sulfate deprived the enzyme of sensitivity to Fru-1,6-P2 or GTP but had no effect on the sensitivity to other effectors. It was found that the activation by the analogs was lost upon desensitization of the enzyme to Fru-1,6-P2 by reaction with 2,4,6-trinitrobenzene sulfonate. The activation by the analogs was not observed in the presence of 200 mM ammonium sulfate. In the presence of lower concentrations (0.1 mM) of PEP, ammonium sulfate activated the enzyme at concentrations less than 700 mM but had an inhibitory effect on the desensitized enzyme. These findings suggest that the unusual phenomena described above are a result of binding of the phosphate esters and sulfate ions with the Fru-1,6-P2 site of the enzyme or the active site depending on the reaction conditions.
Topics: Allosteric Site; Ammonium Sulfate; Carboxy-Lyases; Enzyme Activation; Escherichia coli; Fructosediphosphates; Hexosediphosphates; Phosphoenolpyruvate; Phosphoenolpyruvate Carboxylase; Stereoisomerism; Trinitrobenzenesulfonic Acid
PubMed: 6373747
DOI: 10.1093/oxfordjournals.jbchem.a134652 -
The Journal of Surgical Research Nov 1997Phosphoenolpyruvate (PEP) is a high-energy metabolite in the final step of glycolysis. PEP is converted into pyruvate by pyruvate kinase. One molecule of adenosine...
BACKGROUND
Phosphoenolpyruvate (PEP) is a high-energy metabolite in the final step of glycolysis. PEP is converted into pyruvate by pyruvate kinase. One molecule of adenosine triphosphate (ATP) is generated from one molecule of PEP. The aim of this study was to examine the effects of PEP on hepatic energy metabolism at an early phase after ischemia and reperfusion were examined in rats.
MATERIALS AND METHODS
Male Wistar rats (250-350 g) were divided into two groups; after two 15-min periods of ischemia with 2 min reperfusion in between, either PEP or glucose solution (400 mmol/liter, pH 7.4) was infused into the portal vein (2.5 ml/300 g body wt/5 min). Before and 0, 5, 10, and 30 min after ischemia, arterial blood and liver tissue were collected for analyses.
RESULTS
During the two ischemic periods, ATP and total adenine nucleotide (TAN) of the liver decreased from 9.10 +/- 0.50 and 14.06 +/- 0.29 to 0.99 +/- 0.50 and 10.86 +/- 0.42 mmole/g liver, respectively (P < 0.05), while adenosine monophosphate (AMP) increased from 1.18 +/- 0.15 to 8.47 +/- 0.66 mmole/g liver (P < 0. 05). Hepatic energy charge (EC) significantly decreased from 0.78 +/- 0.02 to 0.16 +/- 0.03 (P < 0.05). Serum concentrations of pyruvate and lactate were elevated from 1.18 +/- 0.15 and 18.4 +/- 0. 52 to 3.29 +/- 0.52 and 72.6 +/- 4.8 mg/dl, respectively (P < 0.05). After a 5-min infusion of PEP or glucose solution, the ATP concentration was significantly higher in the PEP group than in the glucose group (4.08 +/- 0.58 micromole/g liver vs 2.20 +/- 0.45 micromole/g liver, P < 0.01), whereas AMP concentration was significantly lower in the PEP group than in the glucose group (4.26 +/- 0.66 micromole/g liver vs 7.02 +/- 0.71 micromole/g liver, P < 0. 01). EC in the PEP group was significantly higher than that in the glucose group (0.493 +/- 0.051 vs 0.293 +/- 0.042, P < 0.01). Ten minutes after ischemia, the ATP, TAN, and EC levels were still higher in the PEP group than in the glucose group, but the difference did not reach statistical significance. At 30 min after ischemia, these values became similar in both groups. At 5, 10, and 30 min after ischemia, serum pyruvate concentrations were higher in the PEP group than in the glucose group.
CONCLUSION
These findings suggest that PEP recovers hepatic energy from liver cell damage at an early phase after ischemia and reperfusion by prompt ATP production through the degradation of PEP into pyruvate in the liver.
Topics: Adenosine Monophosphate; Adenosine Triphosphate; Animals; Arteries; Energy Metabolism; Infusions, Intravenous; Ischemia; Kinetics; Lactic Acid; Liver; Male; Phosphoenolpyruvate; Portal Vein; Pyruvic Acid; Rats; Rats, Wistar; Reperfusion Injury
PubMed: 9441794
DOI: 10.1006/jsre.1997.5177 -
Research in Microbiology Mar 2007For analyzing the control of energy metabolism in Escherichia coli, we carried out kinetic analyses of glycolytic enzymes purified from the overexpressing clones of E....
For analyzing the control of energy metabolism in Escherichia coli, we carried out kinetic analyses of glycolytic enzymes purified from the overexpressing clones of E. coli K12 W3110 that were constructed with the vector pCA24N. Phosphoenolpyruvate (PEP) acted as an effective inhibitor of enzymes of the preparatory phase in glycolysis. Glucokinase was potently inhibited by PEP in a competitive manner with respect to ATP: the K(i) value for PEP was 0.1mM. PEP further inhibited phosphoglucoisomerase to a lesser extent, and phosphofructokinase A and aldolase A with 10-fold the K(i) values of glucokinase and phosphoglucoisomerase. Glucose is incorporated into E. coli through two pathways: the PTS (PEP-dependent phosphotransferase system) and the glucokinase reaction. PEP, a potent inhibitor of E. coli glucokinase, unlike most eukaryotic hexokinases, can act as a signal molecule controlling glucose uptake and glycolytic flux in cells.
Topics: Escherichia coli K12; Fructose-Bisphosphate Aldolase; Glucokinase; Glycolysis; Phosphoenolpyruvate; Phosphotransferases
PubMed: 17307338
DOI: 10.1016/j.resmic.2006.11.003 -
American Journal of Physiology. Renal... Jun 2023Phosphoenolpyruvate carboxykinase 1 (PCK1 or PEPCK-C) is a cytosolic enzyme converting oxaloacetate to phosphoenolpyruvate, with a potential role in gluconeogenesis,...
Phosphoenolpyruvate carboxykinase 1 (PCK1 or PEPCK-C) is a cytosolic enzyme converting oxaloacetate to phosphoenolpyruvate, with a potential role in gluconeogenesis, ammoniagenesis, and cataplerosis in the liver. Kidney proximal tubule cells display high expression of this enzyme, whose importance is currently not well defined. We generated PCK1 kidney-specific knockout and knockin mice under the tubular cell-specific PAX8 promoter. We studied the effect of PCK1 deletion and overexpression at the renal level on tubular physiology under normal conditions and during metabolic acidosis and proteinuric renal disease. PCK1 deletion led to hyperchloremic metabolic acidosis characterized by reduced but not abolished ammoniagenesis. PCK1 deletion also resulted in glycosuria, lactaturia, and altered systemic glucose and lactate metabolism at baseline and during metabolic acidosis. Metabolic acidosis resulted in kidney injury in PCK1-deficient animals with decreased creatinine clearance and albuminuria. PCK1 further regulated energy production by the proximal tubule, and PCK1 deletion decreased ATP generation. In proteinuric chronic kidney disease, mitigation of PCK1 downregulation led to better renal function preservation. PCK1 is essential for kidney tubular cell acid-base control, mitochondrial function, and glucose/lactate homeostasis. Loss of PCK1 increases tubular injury during acidosis. Mitigating kidney tubular PCK1 downregulation during proteinuric renal disease improves renal function. Phosphoenolpyruvate carboxykinase 1 (PCK1) is highly expressed in the proximal tubule. We show here that this enzyme is crucial for the maintenance of normal tubular physiology, lactate, and glucose homeostasis. PCK1 is a regulator of acid-base balance and ammoniagenesis. Preventing PCK1 downregulation during renal injury improves renal function, rendering it an important target during renal disease.
Topics: Animals; Mice; Acidosis; Glucose; Kidney; Lactates; Mitochondria; Phosphoenolpyruvate; Phosphoenolpyruvate Carboxykinase (GTP)
PubMed: 37102687
DOI: 10.1152/ajprenal.00038.2023 -
Biochemistry Jul 1992In addition to the normal carboxylation reaction, phosphoenolpyruvate carboxylase from Zea mays catalyzes a HCO3(-)-dependent hydrolysis of phosphoenolpyruvate to...
In addition to the normal carboxylation reaction, phosphoenolpyruvate carboxylase from Zea mays catalyzes a HCO3(-)-dependent hydrolysis of phosphoenolpyruvate to pyruvate and Pi. Two independent methods were used to establish this reaction. First, the formation of pyruvate was coupled to lactate dehydrogenase in assay solutions containing high concentrations of L-glutamate and aspartate aminotransferase. Under these conditions, oxalacetic acid produced in the carboxylation reaction was efficiently transaminated, and decarboxylation to form spurious pyruvate was negligible. Second, sequential reduction of oxalacetate and pyruvate was achieved by initially running the reaction in the presence of malate dehydrogenase with NADH in excess over phosphoenolpyruvate. After the reaction was complete, lactate dehydrogenase was added, thus giving a measure of pyruvate concentration. At pH 8.0 in the presence of Mg2+, the rate of phosphoenolpyruvate hydrolysis was 3-7% of the total reaction rate. The hydrolysis reaction catalyzed by phosphoenolpyruvate carboxylase was strongly metal dependent, with rates decreasing in the order Ni2+ greater than Co2+ greater than Mn2+ greater than Mg2+ greater than Ca2+. These results suggest that the active site metal ion binds to the enolate oxygen, thus stabilizing the proposed enolate intermediate. The more stable the enolate, the less reactive it is toward carboxylation and the greater the opportunity for hydrolysis.
Topics: Bicarbonates; Carbon Dioxide; Cations, Divalent; Hot Temperature; Hydrogen-Ion Concentration; Hydrolysis; Phosphoenolpyruvate; Phosphoenolpyruvate Carboxylase; Zea mays
PubMed: 1633156
DOI: 10.1021/bi00143a010