-
Cell Metabolism Nov 2020The mitochondrial GTP (mtGTP)-dependent phosphoenolpyruvate (PEP) cycle couples mitochondrial PEPCK (PCK2) to pyruvate kinase (PK) in the liver and pancreatic islets to...
The mitochondrial GTP (mtGTP)-dependent phosphoenolpyruvate (PEP) cycle couples mitochondrial PEPCK (PCK2) to pyruvate kinase (PK) in the liver and pancreatic islets to regulate glucose homeostasis. Here, small molecule PK activators accelerated the PEP cycle to improve islet function, as well as metabolic homeostasis, in preclinical rodent models of diabetes. In contrast, treatment with a PK activator did not improve insulin secretion in pck2 mice. Unlike other clinical secretagogues, PK activation enhanced insulin secretion but also had higher insulin content and markers of differentiation. In addition to improving insulin secretion, acute PK activation short-circuited gluconeogenesis to reduce endogenous glucose production while accelerating red blood cell glucose turnover. Four-week delivery of a PK activator in vivo remodeled PK phosphorylation, reduced liver fat, and improved hepatic and peripheral insulin sensitivity in HFD-fed rats. These data provide a preclinical rationale for PK activation to accelerate the PEP cycle to improve metabolic homeostasis and insulin sensitivity.
Topics: Animals; Homeostasis; Insulin; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria; Phosphoenolpyruvate; Pyruvate Kinase; Rats; Rats, Sprague-Dawley
PubMed: 33147485
DOI: 10.1016/j.cmet.2020.10.006 -
Journal of Anesthesia Aug 2015Phosphoenolpyruvate (PEP) is an intermediate metabolite of the glycolytic pathway and an in vivo high-energy phosphate compound. We have examined the protective effects...
Phosphoenolpyruvate (PEP) is an intermediate metabolite of the glycolytic pathway and an in vivo high-energy phosphate compound. We have examined the protective effects of PEP on ischemia-reperfusion lung injury in isolated rabbits lungs perfused with a physiological salt solution. The lungs were divided into three treatment groups: (1) ischemia-reperfusion (IR), (2) ischemia-reperfusion with PEP treatment (PEP-IR), in which 1 mM PEP was pre-administered into the perfusate during the stable period, and (3) ventilation-perfusion continued without interruption (Cont). In the IR and PEP-IR groups, ventilation-perfusion was discontinued for about 60 min after a 30-min stable period and then restarted. The capillary filtration coefficients (K fc) and pyruvate concentration in the perfusate were determined immediately before ischemia and 30 and 60 min after reperfusion. The left lungs were dried at the end of the experiment to calculate the tissue wet-to-dry weight ratio (W/D). The K fc values after reperfusion were significantly higher in the IR group than in the other two groups. Pyruvate concentrations were significantly higher at three time-points in the PEP-IR group than in the other two groups. The W/D was significantly higher in the IR group than in the other two groups. Based on these results, we conclude that the administration of PEP prior to lung ischemia alleviates lung ischemia-reperfusion injury.
Topics: Animals; Lung; Lung Diseases; Male; Phosphoenolpyruvate; Rabbits; Reperfusion Injury
PubMed: 25603734
DOI: 10.1007/s00540-014-1972-x -
Molekuliarnaia Genetika, Mikrobiologiia... 1998
Review
Topics: Biological Transport; Carbohydrate Metabolism; Gram-Negative Bacteria; Phosphoenolpyruvate; Phosphotransferases
PubMed: 9819820
DOI: No ID Found -
Archives of Biochemistry and Biophysics Nov 2020A linked-function theory for allostery allows for a differentiation between those protein-ligand interactions that contribute the most to ligand binding and those...
A linked-function theory for allostery allows for a differentiation between those protein-ligand interactions that contribute the most to ligand binding and those protein-ligand interactions that contribute to the allosteric mechanism. This potential distinction is the basis for analogue studies used to determine which chemical moieties on the allosteric effector contribute to allostery. Although less recognized, the same separation of functions is possible for substrate-enzyme interactions. When evaluating allosteric regulation in human liver pyruvate kinase, the use of a range of monovalent cations (K, NH, Rb, Cs, cyclohexylammonium and Tris) altered substrate (phosphoenolpyruvate; PEP) affinity, but maintained similar allosteric responses to the allosteric activator, fructose-1,6-bisphosphate (Fru-1,6-BP). Because crystal structures indicate that the active site monovalent cation interacts directly with the phosphate moiety of the bound PEP substrate, we questioned if the phosphate moiety might contribute to substrate binding, but not to the allosteric mechanism. Here, we demonstrate that the binding of oxalate, a non-phosphorylated substrate/product analogue, is allosterically enhanced by Fru-1,6-BP. That observation is consistent with the concept that the phosphate moiety of PEP is not required for the allosteric function, even though that moiety likely contributes to determining substrate affinity.
Topics: Allosteric Regulation; Fructosediphosphates; Humans; Liver; Phosphoenolpyruvate; Pyruvate Kinase
PubMed: 33075302
DOI: 10.1016/j.abb.2020.108633 -
Biophysical Journal May 2010Structural changes in rabbit muscle pyruvate kinase (PK) induced by phosphoenolpyruvate (PEP) and Mg(2+) binding were studied by attenuated total reflection Fourier...
Structural changes in rabbit muscle pyruvate kinase (PK) induced by phosphoenolpyruvate (PEP) and Mg(2+) binding were studied by attenuated total reflection Fourier transform infrared spectroscopy in combination with a dialysis accessory. The experiments indicated a largely preserved secondary structure upon PEP and Mg(2+) binding but also revealed small backbone conformational changes of PK involving all types of secondary structure. To assess the effect of the protein environment on the bound PEP, we assigned and evaluated the infrared absorption bands of bound PEP. These were identified using 2,3-(13)C(2)-labeled PEP. We obtained the following assignments: 1589 cm(-1) (antisymmetric carboxylate stretching vibration); 1415 cm(-1) (symmetric carboxylate stretching vibration); 1214 cm(-1) (C-O stretching vibration); 1124 and 1110 cm(-1) (asymmetric PO(3)(2-) stretching vibrations); and 967 cm(-1) (symmetric PO(3)(2-) stretching vibration). The corresponding band positions in solution are 1567, 1407, 1229, 1107, and 974 cm(-1). The differences for bound and free PEP indicate specific interactions between ligand and protein. Quantification of the interactions with the phosphate group indicated that the enzyme environment has little influence on the P-O bond strengths, and that the bridging P-O bond, which is broken in the catalytic reaction, is weakened by <3%. Thus, there is only little distortion toward a dissociative transition state of the phosphate transfer reaction when PEP binds to PK. Therefore, our results are in line with an associative transition state. Carboxylate absorption bands indicated a maximal shortening of the length of the shorter C-O bond by 1.3 pm. PEP bound to PK in the presence of the monovalent ion Na(+) exhibited the same band positions as in the presence of K(+), indicating very similar interaction strengths between ligand and protein in both cases.
Topics: Absorption; Animals; Biocatalysis; Enzyme Activation; Magnesium; Phosphoenolpyruvate; Protein Binding; Protein Conformation; Pyruvate Kinase; Rabbits; Sodium; Spectrophotometry, Infrared
PubMed: 20441757
DOI: 10.1016/j.bpj.2009.12.4335 -
Bioresource Technology Jan 2010The uptake and metabolism of sucrose, the major sugar in industrial cane molasses, by Clostridium tyrobutyricum ZJU 8235 was investigated and this study provided the...
The uptake and metabolism of sucrose, the major sugar in industrial cane molasses, by Clostridium tyrobutyricum ZJU 8235 was investigated and this study provided the first definitive evidence for phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) activity in butyric acid-producing bacteria. Glucose was utilized preferentially to sucrose when both substrates were present in the medium. The PEP-dependent sucrose: PTS was induced by growing C. tyrobutyricum on sucrose (but not glucose) as the sole carbon source. Extract fractionation and PTS reconstitution experiments revealed that both soluble and membrane components were required for bioactivity. Sucrose-6-phosphate hydrolase and fructokinase activities were also detected in sucrose-grown cultures. Based on these findings, a pathway of sucrose metabolism in this organism was proposed that includes the forming of sucrose-6-phosphate via the PTS and its further degradation into glucose-6-phosphate and fructose-6-phosphate.
Topics: Biological Transport, Active; Clostridium tyrobutyricum; Enzyme Activation; Models, Biological; Phosphoenolpyruvate; Phosphoenolpyruvate Sugar Phosphotransferase System; Phosphoric Triester Hydrolases; Phosphorylation; Species Specificity; Sucrose
PubMed: 19726178
DOI: 10.1016/j.biortech.2009.08.024 -
Open Biology Mar 2014The inhibition of triosephosphate isomerase (TPI) in glycolysis by the pyruvate kinase (PK) substrate phosphoenolpyruvate (PEP) results in a newly discovered feedback...
The inhibition of triosephosphate isomerase (TPI) in glycolysis by the pyruvate kinase (PK) substrate phosphoenolpyruvate (PEP) results in a newly discovered feedback loop that counters oxidative stress in cancer and actively respiring cells. The mechanism underlying this inhibition is illuminated by the co-crystal structure of TPI with bound PEP at 1.6 Å resolution, and by mutational studies guided by the crystallographic results. PEP is bound to the catalytic pocket of TPI and occludes substrate, which accounts for the observation that PEP competitively inhibits the interconversion of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. Replacing an isoleucine residue located in the catalytic pocket of TPI with valine or threonine altered binding of substrates and PEP, reducing TPI activity in vitro and in vivo. Confirming a TPI-mediated activation of the pentose phosphate pathway (PPP), transgenic yeast cells expressing these TPI mutations accumulate greater levels of PPP intermediates and have altered stress resistance, mimicking the activation of the PK-TPI feedback loop. These results support a model in which glycolytic regulation requires direct catalytic inhibition of TPI by the pyruvate kinase substrate PEP, mediating a protective metabolic self-reconfiguration of central metabolism under conditions of oxidative stress.
Topics: Amino Acid Substitution; Animals; Binding Sites; Biocatalysis; Crystallography, X-Ray; Glyceraldehyde 3-Phosphate; Glycolysis; Humans; Kinetics; Molecular Dynamics Simulation; Phosphoenolpyruvate; Protein Binding; Protein Structure, Tertiary; Rabbits; Recombinant Proteins; Substrate Specificity; Triose-Phosphate Isomerase
PubMed: 24598263
DOI: 10.1098/rsob.130232 -
Canadian Journal of Microbiology Jul 1983Spontaneous mutants defective in some undefined membrane components of the phosphoenolpyruvate:glucose phosphotransferase system were isolated by plating cells of...
Spontaneous mutants defective in some undefined membrane components of the phosphoenolpyruvate:glucose phosphotransferase system were isolated by plating cells of Streptococcus sanguis ATCC 10556 onto an agar containing lactose and 10 mM 2-deoxyglucose. Toluenized cells of these mutants were defective in their ability to catalyse the phosphoenolpyruvate-dependent phosphorylation of 2-deoxyglucose but were still able to phosphorylate alpha-methylglucoside. The phosphorylation of alpha-methylglucoside was essentially dependent on phosphoenolpyruvate and required the presence of both soluble and membrane components. It was concluded that S. sanguis possessed two different phosphoenolpyruvate:glucose phosphotransferase systems.
Topics: Deoxyglucose; Methylglucosides; Methylglycosides; Mutation; Phosphoenolpyruvate; Phosphoenolpyruvate Sugar Phosphotransferase System; Phosphorylation; Streptococcus sanguis
PubMed: 6616345
DOI: 10.1139/m83-136 -
Journal of Biochemistry Feb 1991In the previous paper [Xu, J., Oshima, T., & Yoshida, M. (1990) J. Mol. Biol. 215, 597-606], we reported that phosphofructokinase from Thermus thermophilus is...
In the previous paper [Xu, J., Oshima, T., & Yoshida, M. (1990) J. Mol. Biol. 215, 597-606], we reported that phosphofructokinase from Thermus thermophilus is allosterically inhibited by phosphoenolpyruvate, which induces dissociation of the active four-subunit enzyme into an inactive two-subunit form. When T. thermophilus was cultured in a glucose-containing medium, another phosphofructokinase (PFK2) appeared in addition to the reported one (PFK1). The molecular weights of the native PFK2 molecule (132,000) and its subunit (34,500), which are slightly smaller than those of PFK1, suggest that PFK2 is also composed of four identical subunits. However, the hyperbolic kinetics and molecular form of PFK2 are not affected at all by phosphoenolpyruvate. The NH2-terminal amino acid sequences of subunits of PFK1 and PFK2 revealed that they are composed of very similar but different polypeptides.
Topics: Amino Acid Sequence; Glucose; Isoenzymes; Kinetics; Molecular Sequence Data; Molecular Weight; Phosphoenolpyruvate; Phosphofructokinase-1; Thermus
PubMed: 1830879
DOI: No ID Found -
ACS Chemical Neuroscience Jun 2019Stroke is a leading cause of disability and the second leading cause of death among adults worldwide, while the mechanisms underlying neuronal death and dysfunction...
Stroke is a leading cause of disability and the second leading cause of death among adults worldwide, while the mechanisms underlying neuronal death and dysfunction remain poorly understood. Here, we investigated the differential proteomic profiles of mouse brain homogenate with 3 h of middle cerebral artery occlusion (MCAO) ischemia, or sham, using Coomassie Brilliant Blue staining, followed by mass spectrometry. We identified enolase1 (ENO1), a key glycolytic enzyme, as a potential mediator of neuronal injury in MCAO ischemic model. Reverse transcription polymerase chain reaction and western blotting data showed that ENO1 was ubiquitously expressed in various tissues, distinct regions of brain, and different postnatal age. Immunohistochemical analysis revealed that ENO1 is localized in neuronal cytoplasm and dendrites. Interestingly, the expression level of ENO1 was significantly increased in the early stage, but dramatically decreased in the late stage, of cerebral ischemia in vivo. This dynamic change was consistent with our finding in cultured hippocampal neurons treated with oxygen/glucose deprivation (OGD) in vitro. Importantly, ENO1 overexpression in cultured neurons alleviated dendritic and spinal loss caused by OGD treatment. Furthermore, the enzymatic product of ENO1, phosphoenolpyruvate (PEP), was also synchronously changed along with the dynamic ENO1 level. The neuronal injury caused by OGD treatment in vitro or ischemia in vivo was mitigated by the application of PEP. Taken together, our data revealed that ENO1 plays a novel and protective role in cerebral ischemia-induced neuronal injury, highlighting a potential of ENO1 as a therapeutic target of neuronal protection from cerebral ischemia.
Topics: Animals; Brain Ischemia; HEK293 Cells; Humans; Mice; Neurons; Phosphoenolpyruvate; Phosphopyruvate Hydratase
PubMed: 30943007
DOI: 10.1021/acschemneuro.9b00103