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Journal of Molecular Biology Jan 1994In bacterial phosphofructokinases, either a glutamic or an aspartic residue is present at position 187, and the mechanism of inhibition by phosphoenolpyruvate seems to... (Comparative Study)
Comparative Study
In bacterial phosphofructokinases, either a glutamic or an aspartic residue is present at position 187, and the mechanism of inhibition by phosphoenolpyruvate seems to be correlated to the nature of residue 187. Upon binding phosphoenolpyruvate, only the enzymes with a Glu187 would undergo a major allosteric conformational change from an active into an inactive state, whereas the enzymes with an Asp187 would only show a simple upward shift in their pH-profile of activity. The phosphofructokinase from Spiroplasma citri, which has an Asp187, has been purified and its properties follow this pattern. The behaviour of mutants of the enzyme from Escherichia coli in which Glu187 is replaced by either aspartate or leucine confirms the importance of residue 187. The major allosteric transition of E. coli phosphofructokinase is abolished by the substitution Glu187-->Asp, suggesting that a glutamate at position 187 is necessary (but not sufficient) for the protein to undergo the change from the active into the inactive state induced by phosphenolpyruvate. In addition, the presence of an acidic residue, aspartate or glutamate, at position 187 is required (but not sufficient) for the binding of ADP (or GDP). This requirement of a negative charge for ADP binding could explain the striking conservation of an aspartate residue at position 187 in all the eukaryotic phosphofructokinases.
Topics: Amino Acid Sequence; Bacteria; Glutamates; Glutamic Acid; Humans; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Phosphoenolpyruvate; Phosphofructokinase-1; Saccharomyces cerevisiae; Sequence Homology, Amino Acid; Spiroplasma
PubMed: 7904653
DOI: 10.1016/s0022-2836(05)80014-2 -
Journal of Molecular Biology Jun 1995The fluorescence of the unique tryptophan residue of the allosteric phosphofructokinase from Escherichia coli varies upon binding of any ligand, whether substrate or...
The fluorescence of the unique tryptophan residue of the allosteric phosphofructokinase from Escherichia coli varies upon binding of any ligand, whether substrate or effector, suggesting that the protein undergoes a conformational change. This fluorescent probe has been exploited to determine the rates of the structural transitions that occur upon ligand binding and that are responsible for the remarkable allosteric behavior of this enzyme. The kinetics of fluorescence changes measured after rapidly mixing phosphofructokinase with one of its ligands show the presence of several allosteric transitions with widely different rates, ranging from a few hundred s-1 to less than 0.1 s-1. The rate of each conformational change increases with the concentration of the ligand used to trigger it, suggesting that ligands induce a conformational change and do not displace a pre-existing equilibrium. The hypothesis that each ligand stabilizes a different conformational state of the protein is confirmed by the kinetics of displacement of one ligand by another: for instance, the binary complexes between phosphofructokinase and either its substrate, fructose-6-phosphate, or its allosteric activator, ADP, have the same low fluorescence and should be in the same active state, but they show different rates of conformational transition upon binding the inhibitor phosphoenolpyruvate. It appears that phosphofructokinase can exist in more than two states. Some conformational changes between these multiple states are slow enough to play an important role in the kinetics of the reaction catalyzed by phosphofructokinase, and could even explain part of its allosteric behavior. These results show that steady-state measurements are not sufficient to analyze the regulatory properties of E. coli phosphofructokinase.
Topics: Adenosine Diphosphate; Adenylyl Imidodiphosphate; Allosteric Regulation; Allosteric Site; Crystallography, X-Ray; Escherichia coli; Fructosephosphates; Kinetics; Ligands; Phosphoenolpyruvate; Phosphofructokinase-1; Protein Conformation; Spectrometry, Fluorescence; Time Factors; Tryptophan
PubMed: 7783204
DOI: 10.1006/jmbi.1995.0310 -
Journal of Applied Physiology... May 1995The in vitro activity of skeletal muscle phosphofructokinase (PFK) was determined over the full physiological range of citrate concentrations. Enzyme aggregation was...
The in vitro activity of skeletal muscle phosphofructokinase (PFK) was determined over the full physiological range of citrate concentrations. Enzyme aggregation was enhanced with a crowding agent, as the regulatory properties of PFK are altered with dilution. Cuvette conditions simulated concentrations of effectors and substrates during rest, moderate aerobic exercise, and intense aerobic exercise in human skeletal muscle. Citrate inhibition was not eliminated with enhanced enzyme aggregation, but activity was improved at all citrate concentrations. Maximal PFK activity with no citrate present was 0.27 +/- 0.01 mumol.min-1.mg-1 protein with resting effectors and 1.64 +/- 0.07 and 7.15 +/- 0.52 mumol.min-1.mg-1 protein with moderate aerobic and intense aerobic effector levels, respectively. Under resting conditions, PFK activity decreased to 49% of maximal when citrate was increased from 0 to 0.15 mM and only a small further inhibition to 43% occurred at 0.5 mM. Citrate was a less potent inhibitor under both exercise conditions with the sharpest decline to 72-77% of maximal activity at 0.15 mM followed by a slower decline to 65-70 and 53% activity at 0.25 and 0.5 mM citrate, respectively. The present in vitro measurements predict that alterations in citrate around concentrations normally reported in resting and exercising muscle would have little effect on flux through PFK. Therefore, the generally accepted concept that citrate is a potent inhibitor of PFK in all physiological situations has been exaggerated.
Topics: Animals; Citrates; In Vitro Techniques; Muscle, Skeletal; Phosphofructokinase-1; Physical Exertion; Polyethylene Glycols; Rabbits; Rest
PubMed: 7649922
DOI: 10.1152/jappl.1995.78.5.1853 -
The Journal of Biological Chemistry Nov 1988Phosphofructokinase from the flight muscle of bumblebee was purified to homogeneity and its molecular and catalytic properties are presented. The kinetic behavior...
Phosphofructokinase from bumblebee flight muscle. Molecular and catalytic properties and role of the enzyme in regulation of the fructose 6-phosphate/fructose 1,6-bisphosphate cycle.
Phosphofructokinase from the flight muscle of bumblebee was purified to homogeneity and its molecular and catalytic properties are presented. The kinetic behavior studies at pH 8.0 are consistent with random or compulsory-order ternary complex. At pH 7.4 the enzyme displays regulatory behavior with respect to both substrates, cooperativity toward fructose 6-phosphate, and inhibition by high concentration of ATP. Determinations of glycolytic intermediates in the flight muscle of insects exposed to low and normal temperatures showed statistically significant increases in the concentrations of AMP, fructose 2,6-bisphosphate, and glucose 6-phosphate during flight at 25 degrees C or rest at 5 degrees C. Measuring the activity of phosphofructokinase and fructose 1,6-bisphosphatase at 25 and 7.5 degrees C, in the presence of physiological concentrations of substrates and key effectors found in the muscle of bumblebee kept under different environmental temperatures and activity levels, suggests that the temperature dependence of fructose 6-phosphate/fructose 1,6-bisphosphate cycling may be regulated by fluctuation of fructose 2,6-bisphosphate concentration and changes in the affinity of both enzymes for substrates and effectors. Moreover, in the presence of in vivo concentrations of substrates, phosphofructokinase is inactive in the absence of fructose 2,6-bisphosphate.
Topics: Animals; Bees; Flight, Animal; Fructosediphosphates; Fructosephosphates; Hexosediphosphates; Homeostasis; Kinetics; Muscles; Phosphofructokinase-1; Thermodynamics
PubMed: 2972719
DOI: No ID Found -
The New England Journal of Medicine Oct 1974
Topics: Erythrocytes; Humans; Male; Phosphofructokinase-1
PubMed: 4278172
DOI: 10.1056/nejm197410312911819 -
Biological Chemistry Aug 2013Although the crystal structures of prokaryotic 6-phosphofructokinase, a key enzyme of glycolysis, have been available for almost 25 years now, structural information... (Review)
Review
Although the crystal structures of prokaryotic 6-phosphofructokinase, a key enzyme of glycolysis, have been available for almost 25 years now, structural information about the more complex and highly regulated eukaryotic enzymes is still lacking until now. This review provides an overview of the current knowledge of eukaryotic 6-phosphofructokinase based on recent crystal structures, kinetic analyses and site-directed mutagenesis data with special focus on the molecular architecture and the structural basis of allosteric regulation.
Topics: Allosteric Regulation; Animals; Glycogen Storage Disease Type VII; Glycolysis; Humans; Models, Molecular; Mutation; Phosphofructokinase-1; Protein Conformation
PubMed: 23729568
DOI: 10.1515/hsz-2013-0130 -
The Journal of Biological Chemistry Mar 1987A phosphofructokinase (PFK) has been purified to homogeneity from carrot roots as a large aggregated form (molecular weight greater than 5 million). The purified plant... (Comparative Study)
Comparative Study
A phosphofructokinase (PFK) has been purified to homogeneity from carrot roots as a large aggregated form (molecular weight greater than 5 million). The purified plant PFK, seemingly the cytosolic form, differed from its mammalian counterpart in a lower subunit molecular weight (60,000 verses 80,000), in being only sluggishly activated by fructose-2,6-bisphosphate, and in immunological properties. Similar to liver PFK, the purified carrot PFK could be dissociated by addition of 5 mM ATP to small and intermediate forms (respective molecular mass values of 2.4 X 10(5) and 6 X 10(5) Da). These small and intermediate forms could partially reassociate to the original large form in the presence of 5 mM Fru-6-P. Alkaline pH also effected the dissociation of the large and intermediate forms to the small form of PFK. All forms were present in significant amounts in freshly prepared carrot root extracts. The different forms of PFK showed characteristic pH activity profiles with pH optima of 8.6 (small form), 5.5 and 9.0 (intermediate form), and 7.0 and 8.5 (large forms). As alkaline pH (greater than or equal to approximately 8.5) dissociated the large and intermediate enzyme forms to yield the small form, it was concluded the "true" pH optima of the intermediate and large forms are pH 5.5 and 7.0, respectively. The pH optimum displayed by the intermediate and large forms in the alkaline region (pH 8.5-9.0) was considered to be due to their dissociation during assay. The different forms of PFK also had dissimilar regulatory properties, each showing a characteristic response to ATP, citrate, and Pi, but all were sensitive to inhibition by phosphoenolpyruvate and NADPH. Leaf cytosolic PFK, partially purified from spinach, showed similar properties. The results suggest that metabolite-dependent aggregation-disaggregation is a mechanism whereby plants regulate the activity of cytosolic PFK and the accompanying rate of glycolytic carbon flux.
Topics: Adenosine Triphosphate; Cytosol; Enzyme Activation; Epitopes; Fructosediphosphates; Fructosephosphates; Hydrogen-Ion Concentration; Liver; Macromolecular Substances; Molecular Weight; NADP; Phosphoenolpyruvate; Phosphofructokinase-1; Plants; Vegetables
PubMed: 2434498
DOI: No ID Found -
Proceedings of the National Academy of... Oct 1980Pure phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) from liver is strongly inhibited by ATP, whereas crude phosphofructokinase is...
Pure phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) from liver is strongly inhibited by ATP, whereas crude phosphofructokinase is only slightly inhibited by ATP. A factor that is removed from the enzyme during purification and can prevent the inhibition of phosphofructokinase by ATP has been isolated. The factor can be resolved into three components that differ in molecular weights, as shown by gel filtration on Sephadex G-25. These factors overcome the ATP inhibition but have no effect on the catalytic activity under the optimum assay conditions. Furthermore, AMP acts syngeristically with the activation factor in reversing ATP inhibition. It is proposed that the activation of phosphofructokinase by the activation factor and AMP is sufficient to account for the glycolytic flux in the liver.
Topics: Adenosine Triphosphate; Animals; Enzyme Activation; Kinetics; Liver; Phosphofructokinase-1; Rats
PubMed: 6449699
DOI: 10.1073/pnas.77.10.5861 -
Increased phosphofructokinase content during chronic hypoxia in cultured skeletal muscle (L8) cells.Biochimica Et Biophysica Acta Sep 1983Chronic hypoxia results in increased measured activity of all of the glycolytic enzymes and is associated with an increase in glycolytic capacity. Phosphofructokinase, a...
Chronic hypoxia results in increased measured activity of all of the glycolytic enzymes and is associated with an increase in glycolytic capacity. Phosphofructokinase, a rate-limiting glycolytic enzyme, was measured under normoxic and hypoxic conditions to determine the relationship between increased activity and enzyme content. Monoclonal antibodies were used to isolate pure enzyme in rat skeletal muscle cells (L8) cultured hypoxically (PO2 = 14 torr) and normoxically (PO2 = 142 torr). Phosphofructokinase content per cell in cultures maintained under chronic (96 h) hypoxic conditions was twice that of cells cultured under normoxic conditions (0.0675 +/- 0.008 (S.E.) and 0.0345 +/- 0.003 micrograms enzyme protein/microgram DNA, P less than 0.01). Phosphofructokinase activity increased proportionately (hypoxia, 0.020 +/- 0.003; normoxia, 0.010 +/- 0.001 units/microgram DNA). The specific activity (units/mg enzyme protein) of phosphofructokinase in the hypoxic (296 +/- 32) versus the normoxic (290 +/- 15) cultures was not significantly different, indicating that the increased activity was accounted for by an increase in enzyme content. Glycolytic rate appears to be regulated at the level of enzyme content.
Topics: Animals; Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; Cells, Cultured; Enzyme-Linked Immunosorbent Assay; Hypoxia; Muscles; Phosphofructokinase-1; Radioimmunoassay; Rats
PubMed: 6225466
DOI: 10.1016/0167-4889(83)90040-x -
Methods in Enzymology 1975
Topics: Ammonium Sulfate; Calcium Phosphates; Chromatography, Gel; Enterobacter; Fractional Precipitation; Gels; Hydrogen-Ion Concentration; Kinetics; Magnesium; Methods; Molecular Weight; Phosphofructokinase-1; Protamines; Spectrophotometry
PubMed: 237207
DOI: 10.1016/0076-6879(75)42094-8