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FEBS Letters Sep 1999We have determined the crystal structure of Mn2+-bound Escherichia coli phosphoenolpyruvate carboxylase (PEPC) using X-ray diffraction at 2.6 A resolution, and specified...
We have determined the crystal structure of Mn2+-bound Escherichia coli phosphoenolpyruvate carboxylase (PEPC) using X-ray diffraction at 2.6 A resolution, and specified the location of enzyme-bound Mn2+, which is essential for catalytic activity. The electron density map reveals that Mn2+ is bound to the side chain oxygens of Glu-506 and Asp-543, and located at the top of the alpha/beta barrel in PEPC. The coordination sphere of Mn2+ observed in E. coli PEPC is similar to that of Mn2+ found in the pyruvate kinase structure. The model study of Mn2+-bound PEPC complexed with phosphoenolpyruvate (PEP) reveals that the side chains of Arg-396, Arg-581 and Arg-713 could interact with PEP.
Topics: Animals; Aspartic Acid; Binding Sites; Catalysis; Computer Simulation; Crystallography, X-Ray; Escherichia coli; Manganese; Models, Molecular; Peptide Fragments; Phosphoenolpyruvate; Phosphoenolpyruvate Carboxylase; Protein Structure, Secondary; Pyruvate Kinase; Rabbits; Sequence Homology, Amino Acid
PubMed: 10481043
DOI: 10.1016/s0014-5793(99)01103-5 -
Journal of Biochemistry May 1977Accumulation of exogenous phosphoenolpyruvate against the concentration gradient was observed when human red cells were incubated in an acidified isotonic sucrose...
Accumulation of exogenous phosphoenolpyruvate against the concentration gradient was observed when human red cells were incubated in an acidified isotonic sucrose medium. Fluoride increased the apparent accumulation by inhibition of the intracellular metabolic interconversion of the phosphate compound. The accumulation appeared to be specific for phosphoenolpyruvate and the accumulation rate for 3-phosphoglycerate, which has a molecular size and pKa similar to those of phosphoenolpyruvate, was less than one-tenth of the rate of phosphoenolpyruvate. Red cells incubated in the acidified sucrose medium tended to adhere to each other when examined with a scanning electron microscope.
Topics: Biological Transport, Active; Erythrocytes; Fluorides; Humans; Hydrogen-Ion Concentration; Kinetics; Microscopy, Electron, Scanning; Osmolar Concentration; Phosphoenolpyruvate; Sucrose
PubMed: 19438
DOI: No ID Found -
Tanpakushitsu Kakusan Koso. Protein,... 1977
Topics: Allosteric Regulation; Carboxy-Lyases; Chemical Phenomena; Chemistry; Phosphoenolpyruvate
PubMed: 611561
DOI: No ID Found -
Biochimica Et Biophysica Acta Feb 1976Phosphoenolpyruvate was found to depress extra oxygen consumption associated with Ca2+ -induced respiratory jump by rat heart mitochondria. Addition of...
Phosphoenolpyruvate was found to depress extra oxygen consumption associated with Ca2+ -induced respiratory jump by rat heart mitochondria. Addition of phosphoenolpyruvate to mitochondria which have accumulated Ca2+ in the presence of glutamate and inorganic phosphate causes the release of Ca2+ from mitochondria. The phosphoenolpyruvate-stimulated Ca2+ efflux can be observed with mitochondria loaded with low initial Ca2+ concentration (0.12 mM) in the incubation medium. Measurements of mitochondrial H+ translocation produced by addition of Ca2+ to respiring mitochondria show that phosphoenolpyruvate depresses H+ ejection and enhances H+ uptake by mitochondria. The Ca2+ -releasing effect of phosphoenolpyruvate was found to be significantly stronger than that produced by rotenone when added to mitochondria loaded with Ca2+ in the presence of glutamate and inorganic phosphate. Dithiothreitol cannot overcome the effect of phosphoenolpyruvate on mitochondrial Ca2+ transport.
Topics: Animals; Biological Transport, Active; Calcium; Mitochondria, Muscle; Myocardium; Oxygen Consumption; Phosphoenolpyruvate; Rats; Rotenone
PubMed: 1247607
DOI: 10.1016/0005-2728(76)90178-x -
The Thoracic and Cardiovascular Surgeon Apr 1986The paracorporeal rat heart model was used to investigate the extent of myocardial protection by potassium cardioplegia supplemented with phosphoenolpyruvate (PEP) (14.4...
The paracorporeal rat heart model was used to investigate the extent of myocardial protection by potassium cardioplegia supplemented with phosphoenolpyruvate (PEP) (14.4 mM) and ATP (0.067 mM) singly or in combination. Rat hearts were subjected to 30 minutes of ischemia at 37 degrees. They were subsequently reperfused for 40 minutes during which time the left ventricular isovolumic work was measured and blood samples were taken for creatine kinase isoenzyme MB (CK-MB) analysis. At the end of each experiment the hearts were freeze-clamped for later analyses of high energy phosphate compounds. Supplementation with PEP and ATP (Group I) and with only ATP (Group II) showed a significantly better left ventricular isovolumic work and a significantly higher adenylate charge potential (ACP). Supplementation with only PEP (Group III) resulted in significantly better left ventricular isovolumic work than the control group but significantly lower than groups I and II. There were no significant differences between the groups in regard to the CK-MB efflux. Supplementation with PEP and ATP in combination did not show any positive effect at 40 minutes of reperfusion over and above that which was achieved with ATP only.
Topics: Adenosine Triphosphate; Animals; Creatine Kinase; Heart; Heart Arrest, Induced; Heart Ventricles; Isoenzymes; Male; Myocardium; Phosphoenolpyruvate; Rats; Rats, Inbred Strains
PubMed: 2424124
DOI: 10.1055/s-2007-1020388 -
Biochimie 1976The phosphocarrier protein (HPr) of the phosphoenol pyruvate : alpha-methyl-D-glucoside-phosphotransferase system (PTS) has been purified from Bacillus subtilis Marburg...
The phosphoenolpyruvate : methyl-alpha-D-glucoside phosphotransferase system in Bacillus subtilis Marburg 168 : purification and identification of the phosphocarrier protein (HPr).
The phosphocarrier protein (HPr) of the phosphoenol pyruvate : alpha-methyl-D-glucoside-phosphotransferase system (PTS) has been purified from Bacillus subtilis Marburg 168. The molecular weight is about 8300. HPr contains 1 histidine residue. Phophoenzyme I appears to be an intermediate in the initial phosphoryl transfer from phosphoenolpyruvate (PEP) to HPr. Phospho-HPr is isolated and characterized as a component of the complete system.
Topics: Bacillus subtilis; Carrier Proteins; Histidine; Isoelectric Focusing; Methylglucosides; Multienzyme Complexes; Peptide Fragments; Phosphoenolpyruvate; Phosphotransferases
PubMed: 820382
DOI: 10.1016/s0300-9084(76)80254-4 -
Response of phosphoenolpyruvate cycle activity to fasting and to hyperinsulinemia in human subjects.The American Journal of Physiology Jul 1996We have used a new isotopic tracer technique to investigate the physiological role of the phosphoenolpyruvate (PEP) cycle in metabolic adaptation to fasting and to...
We have used a new isotopic tracer technique to investigate the physiological role of the phosphoenolpyruvate (PEP) cycle in metabolic adaptation to fasting and to hyperinsulinemia. The forward direction of the PEP cycle is the conversion of oxaloacetate (OAA) to PEP, and the net flux of the cycle is the rate at which PEP from OAA goes on to form glucose or glycogen, as opposed to being recycled to pyruvate and then OAA. Normal volunteers (n = 6) were studied after an overnight fast and then again after 3 days of fasting, and five additional subjects were studied during a hyperinsulinemic clamp (insulin concentration = 568 +/- 25 microU/ml, glucose infusion = 14.2 +/- 0.55 mg.kg-1.min-1). After an overnight fast, 35.4 +/- 6.7% of PEP from OAA was recycled to pyruvate-lactate. Short-term fasting caused a significant increase in the conversion of OAA to PEP and also a drop in the percentage of PEP from OAA that went to pyruvate-lactate to 15.2 +/- 4.0%. The principal response to hyperinsulinemia was a decrease in the recycling of OAA to lactate, because there was no significant change in the conversion of OAA to PEP. We conclude that changes in both directions of the PEP cycle are important in regulating gluconeogenic-glyconeogenic flux.
Topics: Fasting; Humans; Hyperinsulinism; Lactic Acid; Male; Models, Biological; Oxaloacetates; Phosphoenolpyruvate; Pyruvic Acid
PubMed: 8760094
DOI: 10.1152/ajpendo.1996.271.1.E159 -
Metabolic Engineering Oct 2001Metabolic engineering to design and construct microorganisms suitable for the production of aromatic amino acids and derivatives thereof requires control of a... (Review)
Review
Metabolic engineering to design and construct microorganisms suitable for the production of aromatic amino acids and derivatives thereof requires control of a complicated network of metabolic reactions that partly act in parallel and frequently are in rapid equilibrium. Engineering the regulatory circuits, the uptake of carbon, the glycolytic pathway, the pentose phosphate pathway, and the common aromatic amino acid pathway as well as amino acid importers and exporters that have all been targeted to effect higher productivities of these compounds are discussed.
Topics: Amino Acids, Aromatic; Biological Transport; Escherichia coli; Feedback; Genetic Engineering; Phosphoenolpyruvate; Sugar Phosphates
PubMed: 11676565
DOI: 10.1006/mben.2001.0196 -
Biochemistry Dec 1985(E)-3-Cyanophosphoenolpyruvate has been synthesized by reacting dimethyl chlorophosphate with the potassium enolate of ethyl cyanopyruvate. The resulting trialkyl ester...
(E)-3-Cyanophosphoenolpyruvate has been synthesized by reacting dimethyl chlorophosphate with the potassium enolate of ethyl cyanopyruvate. The resulting trialkyl ester was deesterified with bromotrimethylsilane followed by potassium hydroxide. Subsequent treatment with Dowex-50-H+ resin and cyclohexylamine afforded the tricyclohexylammonium salt; only the E geometric isomer was obtained. This compound can be photoisomerized to a 70:30 E:Z mixture. (E)-3-Cyanophosphoenolpyruvate is an excellent competitive inhibitor of phosphoenolpyruvate carboxylase [KI(Mn2+) = 16 microM, KI(Mg2+) = 1360 microM], pyruvate kinase [KI(Mn2+) = 0.085 microM, KI(Mg2+) = 0.76 microM], and enolase [KI(Mn2+) = 360 microM, KI(Mg2+) = 280 microM]. The compound is a substrate for pyruvate kinase (Vmax approximately 1% of phosphoenolpyruvate rate), but not for the other two enzymes. No irreversible inactivation is observed with phosphoenolpyruvate carboxylase of pyruvate kinase.
Topics: Animals; Binding, Competitive; Enzyme Inhibitors; In Vitro Techniques; Kinetics; Phosphoenolpyruvate; Phosphoenolpyruvate Carboxylase; Phosphopyruvate Hydratase; Pyruvate Kinase; Rabbits; Substrate Specificity
PubMed: 4092027
DOI: 10.1021/bi00347a015 -
Comparative Biochemistry and... Nov 1997Enterocytes from fasted rabbits make glucose from exogenous fructose and dihydroxyacetone at rates of 180 and 91 nmol/min/10(8) cells but do not make glucose from...
Enterocytes from fasted rabbits make glucose from exogenous fructose and dihydroxyacetone at rates of 180 and 91 nmol/min/10(8) cells but do not make glucose from glycerol, aspartate, malate, lactate, alpha-ketoglutarate, glutamate or glutamine. Total activities of phosphoenolpyruvate carboxykinase, fructose 1,6-bisphosphatase and glucose 6-phosphatase in isolated enterocytes are 0.44, 0.60 and 1.90 mumol/min/10(8) cells, and > or = 95% of carboxykinase activity is intramitochondrial. Enterocytes contain marginal glycerol kinase (0.05 mumol/ min/10(8) cells) and essentially no pyruvate carboxylase activities. Enterocyte mitochondria synthesize citrate from exogenous phosphoenolpyruvate and acetylcarnitine at a rate of 2.40 nmol/min/mg protein. Citrate formation is highly dependent on exogenous HCO3 and inhibited strongly by 3-mercaptopicolinate and 1,2,3-benzenetricarboxylate. Citrate synthesis is stimulated consistently by GDP and significantly so by GTP. Citrate production is unaffected by ADP or ATP. Enterocytes from fasted-refed rabbits contain activities of 0.05, 0.12, 0.39 and 0.56 mumol/min/mg cytosolic protein of ATP:citrate lyase, NADP:malate dehydrogenase, glucose 6-phosphate dehydrogenase and NADP:isocitrate dehydrogenase. Activities of NADP:malate dehydrogenase, glucose 6-phosphate dehydrogenase and NADP:isocitrate dehydrogenase are significantly higher in enterocytes from fasted-refed rabbits than those from fasted rabbits. Mitochondrial phosphoenolpyruvate carboxykinase in enterocytes in vivo could convert glycolysis-derived phosphoenolpyruvate to oxaloacetate that, with acetyl CoA, could form citrate for export to support cytosolic lipogenesis as an activator of acetyl CoA carboxylase, a source of carbon via ATP:citrate lyase and of NADPH via NADP:malate dehydrogenase or NADP:isocitrate dehydrogenase.
Topics: Acetylcarnitine; Animals; Citric Acid; Female; Glucose; Intestine, Small; Male; Mitochondria; Phosphoenolpyruvate; Rabbits
PubMed: 9467872
DOI: 10.1016/s0305-0491(97)00242-3