-
Journal of Bacteriology May 1977In the absence of an exogenous energy source, galactose-grown cells of Streptococcus lactis ML3 rapidly accumulated thiomethyl-beta-D-galactopyranoside (TMG) and...
In the absence of an exogenous energy source, galactose-grown cells of Streptococcus lactis ML3 rapidly accumulated thiomethyl-beta-D-galactopyranoside (TMG) and 2-deoxyglucose to intracellular concentrations of 40 to 50 mM. Starved cells maintained the capacity for TMG uptake for many hours, and accumulation of the beta-galactoside was insensitive to proton-conducting ionophores (tetrachlorosalicylanilide and carbonylcyanide-m-chlorophenyl hydrazone) and sulfydryl group reagents including iodoacetate and N-ethylmaleimide. Fluorimetric analysis of glycolytic intermediates in extracts prepared from starved cells revealed (a) high intracellular levels of phosphoenolpyruvate (13 mM; PEP) and 2-phosphoglycerate (approximately 39 mM; 2-PG), but an absence of other metabolites including glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-diphosphate, and triosephosphates. The following criteria showed PEP (and 2-PG) to be the endogenous energy source for TMG accumulation by the phosphotransferase system: the intracellular concentrations of PEP and 2-PG decreased with concomitant uptake of TMG, and a close correlation was observed between maximum accumulation of the beta-galactoside and the total available concentration of the two intermediates; TMG accumulated as an anionic derivative, which after extraction and incubation with alkaline phosphatase (EC 3.1.3.1) formed the original analogue; fluoride inhibition of 2-phospho-D-glycerate hydrolyase (EC 4.2.1.11) prevented the conversion of 2-PG to PEP, and uptake of TMG by the starved cells was reduced by 80%; and the stoichiometric ratio [TMG] accumulated/[PEP] consumed was almost unity (0.93). In cells metabolizing glucose, all intermediates listed in (a) and (b) were found. Upon exhaustion of glucose from the medium, the metabolites in (b) were not longer detectable, while the intracellular concentrations of PEP and 2-PG increased to the levels previously observed in starved cells. The glycolytic intermediates in (b) are all in vitro heterotropic effectors of pyruvate kinase (adenosine 5'-triphosphate:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from S. lactis ML3. It is suggested that the capacity of starved cells to maintain high intracellular concentrations of PEP and 2-PG is a consequence of decreased in vivo activity of this key regulatory enzyme of glycolysis.
Topics: Biological Transport; Carbohydrate Metabolism; Carbon Radioisotopes; Energy Metabolism; Fluorides; Glyceric Acids; Glycolysis; Kinetics; Lactococcus lactis; Phosphoenolpyruvate; Time Factors; Tritium
PubMed: 122509
DOI: 10.1128/jb.130.2.583-595.1977 -
Biochemistry Feb 1984The halogenated phosphoenolpyruvate analogues (Z)-phosphoenol-3-fluoropyruvate, (E)-phosphoenol-3-fluoropyruvate, and (Z)-phosphoenol-3-bromopyruvate were synthesized... (Comparative Study)
Comparative Study
The halogenated phosphoenolpyruvate analogues (Z)-phosphoenol-3-fluoropyruvate, (E)-phosphoenol-3-fluoropyruvate, and (Z)-phosphoenol-3-bromopyruvate were synthesized and purified. The analogues were characterized by 1H and by 19F NMR where applicable. Absolute stereoselectivity of the fluorophosphoenolpyruvate isomers as substrates with the enzymes phosphoenolpyruvate carboxykinase, enolase, and pyruvate phosphate dikinase was observed. The Z isomer exhibited substrate activity with these enzymes while no substrate activity was measured with the E isomer. Both isomers exhibited substrate activity with the enzyme pyruvate kinase, however, with a substantial decrease in the Vmax/Km ratio compared to phosphoenolpyruvate as the substrate. A metal ion dependent stereoselectivity of inhibition was measured for these analogues with the enzymes phosphoenolpyruvate carboxykinase, enolase, and pyruvate kinase. The cation activator appears to affect the specificity and thus the catalytic site of these enzymes. Proton longitudinal relaxation rate titrations demonstrate that the dissociation constants, K3, of the fluorophosphoenolpyruvate isomers from the enzyme-Mn complex agree, in most cases, with the measured KI values and analogue binding resembles phosphoenolpyruvate binding. With the enzyme phosphoenolpyruvate carboxykinase, the KI not equal to K3 for (E)-fluorophosphoenolpyruvate which suggests that the binding of the E isomer is affected by the presence of the other substrates. The halogenated derivatives apparently undergo an enzyme-Mn catalyzed Michael-type addition reaction with the bromo-substituted analogue decomposing much faster than the fluoro analogues.
Topics: Animals; Kinetics; Phosphoenolpyruvate; Phosphoenolpyruvate Carboxykinase (GTP); Phosphopyruvate Hydratase; Pyruvate Kinase; Rabbits; Stereoisomerism; Substrate Specificity
PubMed: 6712918
DOI: 10.1021/bi00299a012 -
The Biochemical Journal Jun 2020Activation of phosphoenolpyruvate carboxylase (PEPC) enzymes by glucose 6-phosphate (G6P) and other phospho-sugars is of major physiological relevance. Previous kinetic,...
Activation of phosphoenolpyruvate carboxylase (PEPC) enzymes by glucose 6-phosphate (G6P) and other phospho-sugars is of major physiological relevance. Previous kinetic, site-directed mutagenesis and crystallographic results are consistent with allosteric activation, but the existence of a G6P-allosteric site was questioned and competitive activation-in which G6P would bind to the active site eliciting the same positive homotropic effect as the substrate phosphoenolpyruvate (PEP)-was proposed. Here, we report the crystal structure of the PEPC-C4 isozyme from Zea mays with G6P well bound into the previously proposed allosteric site, unambiguously confirming its existence. To test its functionality, Asp239-which participates in a web of interactions of the protein with G6P-was changed to alanine. The D239A variant was not activated by G6P but, on the contrary, inhibited. Inhibition was also observed in the wild-type enzyme at concentrations of G6P higher than those producing activation, and probably arises from G6P binding to the active site in competition with PEP. The lower activity and cooperativity for the substrate PEP, lower activation by glycine and diminished response to malate of the D239A variant suggest that the heterotropic allosteric activation effects of free-PEP are also abolished in this variant. Together, our findings are consistent with both the existence of the G6P-allosteric site and its essentiality for the activation of PEPC enzymes by phosphorylated compounds. Furthermore, our findings suggest a central role of the G6P-allosteric site in the overall kinetics of these enzymes even in the absence of G6P or other phospho-sugars, because of its involvement in activation by free-PEP.
Topics: Allosteric Regulation; Catalytic Domain; Glucose-6-Phosphate; Kinetics; Phosphoenolpyruvate; Phosphoenolpyruvate Carboxylase; Plant Proteins; Zea mays
PubMed: 32459324
DOI: 10.1042/BCJ20200304 -
Cell Cycle (Georgetown, Tex.) Nov 2010
Topics: Adenosine Triphosphate; Citric Acid Cycle; Energy Metabolism; Glycolysis; Humans; Neoplasms; Phosphoenolpyruvate; Phosphoglycerate Mutase; Protein Isoforms; Pyruvate Kinase
PubMed: 21045562
DOI: 10.4161/cc.9.21.13925 -
Infection and Immunity Jun 1979A phosphoenolpyruvate-dependent sucrose phosphotransferase system has been identified in Streptococcus mutans. Sucrose phosphotransferase activity was inducible by...
A phosphoenolpyruvate-dependent sucrose phosphotransferase system has been identified in Streptococcus mutans. Sucrose phosphotransferase activity was inducible by sucrose and had an apparent Km for sucrose of 70 microM. The product of the sucrose phosphotransferase reaction was isolated and identified as sucrose phosphate. Additional analysis revealed that the phosphate group was on the glucose moiety. Mutants unable to grow in media containing low concentrations of sucrose were isolated and found to be missing either sucrose phosphotransferase activity or the ability to hydrolyze sucrose phosphate.
Topics: Enzyme Induction; Kinetics; Mutation; Phosphoenolpyruvate; Phosphotransferases; Streptococcus mutans; Sucrose; Sugar Phosphates
PubMed: 468378
DOI: 10.1128/iai.24.3.865-868.1979 -
Molekuliarnaia Genetika, Mikrobiologiia... Feb 1991The data of foreign researchers on the specific phosphoenolpyruvate-dependent phosphotransferase systems (PTS) transporting mannose, aminosugars and natural... (Review)
Review
The data of foreign researchers on the specific phosphoenolpyruvate-dependent phosphotransferase systems (PTS) transporting mannose, aminosugars and natural beta-glucosides are reviewed. The genetical, biochemical and molecular biological aspects of the corresponding PTS functioning are presented. The role of those PTS in bacteriophage lambda DNA penetration into the cell, in streptozotocin resistance is discussed.
Topics: Bacteriophage lambda; Biological Transport; Carbohydrate Metabolism; DNA, Viral; Enterobacteriaceae; Phosphoenolpyruvate
PubMed: 1827656
DOI: No ID Found -
International Journal of Molecular... Nov 2021Some metabolic pathways involve two different cell components, for instance, cytosol and mitochondria, with metabolites traffic occurring from cytosol to mitochondria... (Review)
Review
Some metabolic pathways involve two different cell components, for instance, cytosol and mitochondria, with metabolites traffic occurring from cytosol to mitochondria and vice versa, as seen in both glycolysis and gluconeogenesis. However, the knowledge on the role of mitochondrial transport within these two glucose metabolic pathways remains poorly understood, due to controversial information available in published literature. In what follows, we discuss achievements, knowledge gaps, and perspectives on the role of mitochondrial transport in glycolysis and gluconeogenesis. We firstly describe the experimental approaches for quick and easy investigation of mitochondrial transport, with respect to cell metabolic diversity. In addition, we depict the mitochondrial shuttles by which NADH formed in glycolysis is oxidized, the mitochondrial transport of phosphoenolpyruvate in the light of the occurrence of the mitochondrial pyruvate kinase, and the mitochondrial transport and metabolism of L-lactate due to the L-lactate translocators and to the mitochondrial L-lactate dehydrogenase located in the inner mitochondrial compartment.
Topics: Animals; Biological Transport; Gluconeogenesis; Glycolysis; Humans; Mitochondria; NAD; Phosphoenolpyruvate; Pyruvate Kinase
PubMed: 34884425
DOI: 10.3390/ijms222312620 -
Archives of Biochemistry and Biophysics Jan 1976
Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Atractyloside; Biological Transport, Active; Calcium; Coenzyme A; Guinea Pigs; Kinetics; Male; Mitochondria, Muscle; Mitochondrial ADP, ATP Translocases; Myocardium; Nucleotidyltransferases; Phosphoenolpyruvate
PubMed: 1252077
DOI: 10.1016/0003-9861(76)90071-0 -
Analytical Biochemistry Jan 1983
Comparative Study
Topics: Chromatography, DEAE-Cellulose; Escherichia coli; Phosphoenolpyruvate; Phosphoenolpyruvate Carboxykinase (GTP)
PubMed: 6342465
DOI: 10.1016/0003-2697(83)90372-x -
Infection and Immunity Jun 1979A phosphoenolpyruvate-dependent sucrose phosphotransferase system (PTS) has been demonstrated, by an enzyme-coupled reaction and product isolation, in decryptified cell...
A phosphoenolpyruvate-dependent sucrose phosphotransferase system (PTS) has been demonstrated, by an enzyme-coupled reaction and product isolation, in decryptified cell suspensions of the cariogenic microorganism Streptococcus mutans NCTC 10449. The apparent sucrose PTS reaction for sucrose-adapted, sucrose-challenged cells displayed saturation kinetics with an apparent Km of 7.14 x 10(-5) M, which was distinct from the Km of the glucose PTS activity of glucose-adapted, glucose-challenged cells. Both the sucrose and the glucose PTS activities appear to be inducible and under separate genetic control. The sucrose PTS reaction demonstrated in decryptified cells had an absolute requirement for phosphoenolpyruvate. Only 2-phosphoglycerate, the immediate glycolytic precursor of phosphoenolpyruvate, was found to substitute for phosphoenolpyruvate in this reaction in the absence of fluoride. The sucrose PTS activity of sucrose-adapted cells was competitively inhibited by raffinose and lactose; these same sugars had no effect on the apparent glucose PTS activity. Fructose was the only carbohydrate tested other than sucrose which elicited an apparent PTS reaction in sucrose-adapted cells. The product of the sucrose PTS reaction was isolated and behaved chromatographically on a Dowex-1-X8 column like a monophosphate ester. Alkaline phosphatase treatment of the presumptive sucrose monophosphate liberated a component which behaved chromatographically like free sucrose. Subsequent acid hydrolysis of this component produced moieties which behaved chromatographically like glucose and fructose.
Topics: Enzyme Induction; Fructose; Glucose; Kinetics; Lactose; Phosphoenolpyruvate; Phosphotransferases; Raffinose; Streptococcus mutans; Sucrose
PubMed: 468377
DOI: 10.1128/iai.24.3.821-828.1979