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Biochemical Society Transactions Aug 1990
Topics: Adenosine Triphosphate; Animals; Citrates; Citric Acid; Enzyme Activation; Hexosediphosphates; Kinetics; Muscles; Phosphofructokinase-1; Rana temporaria
PubMed: 2148917
DOI: 10.1042/bst0180592 -
The Journal of Biological Chemistry Nov 1975A pyrophosphate-dependent phosphofructokinase (pyrophosphate; D-fructose-6-phosphate-1-phosphotransferase) has been purified and characterized from extracts of...
A pyrophosphate-dependent phosphofructokinase (pyrophosphate; D-fructose-6-phosphate-1-phosphotransferase) has been purified and characterized from extracts of Propionibacterium shermanii. The enzyme catalyzes the transfer of phosphate from pyrophosphate to fructose 6-phosphate to yield fructose-1,6-P2 and phosphate. This unique enzymatic activity was observed initially in Entamoeba histolytica (Reeves, R.E., South, D.J., Blytt, H.G., and Warren, L. G. (1974) J. Biol. Chem. 249, 7734-7741). This is the third pyrophosphate-utilizing enzyme that these two diverse organisms have in common. The others are phosphoenolpyruvate carboxytransphosphorylase and pyruvate phosphate dikinase. The PPi-phosphofructokinase from P. shermanii is specific for fructose-6-P and fructose-1,6-P2, no other phosphorylated sugars were utilized. Phosphate could be replaced by arsenate. The Km values are: phosphate, 6.0 X 10(-4) M; fructose-1, 6-P2, 5.1 X 10(-5) M; pyrophosphate, 6.9 X 10(-5) M; and fructose-6-P, 1.0 X 10(-4) M. The S20w is 5.1 S. The molecular weight of the native enzyme is 95,000. Sodium dodecyl sulfate electrophoresis of the enzyme showed a single band migrating with an Rf corresponding to a molecular weight of 48,000. Extracts of P. shermanii have PPi-phosphofructokinase activity approximately 6 times greater than ATP-phosphofructokinase and 15 to 20 times greater than fructose diphosphatase activities. It is proposed that (a) PPi may replace ATP in the formation of fructose-1-6-P2 when the organism is grown on glucose and (b) when the organism is grown on lactate or glycerol the conversion of fructose-1,6-P2 to fructose-6-P during gluconeogenesis may occur by phosphorolysis rather than hydrolysis.
Topics: Diphosphates; Enzyme Activation; Kinetics; Magnesium; Manganese; Molecular Weight; Phosphofructokinase-1; Propionibacterium; Structure-Activity Relationship
PubMed: 171261
DOI: No ID Found -
Proceedings of the National Academy of... Dec 1986Microinjection of muscle 6-phosphofructokinase (PFK; EC 2.7.1.11) into tissue culture cells led to a reversible disintegration of microfilament bundles (stress fibers)....
Microinjection of muscle 6-phosphofructokinase (PFK; EC 2.7.1.11) into tissue culture cells led to a reversible disintegration of microfilament bundles (stress fibers). The mode of disruption as well as of recovery of stress fibers was very similar to that found previously in experiments performed with the actin-severing protein brevin, an extracellular variant of gelsolin. PFK, like brevin, was also capable of disrupting stress fibers in detergent-extracted cells and in ethanol-fixed cells, in a Ca2+-dependent manner. When compared with heart muscle gelsolin, PFK comigrated with the 85- to 90-kDa band. Antibodies against PFK crossreacted with gelsolin from the same species. These results point to a tight association between polypeptides with similar biochemical and immunological parameters present in both preparations. They suggest hitherto unexpected cellular control mechanisms for both microfilament functions and glycolysis.
Topics: Actin Cytoskeleton; Animals; Calcium-Binding Proteins; Cell Line; Chick Embryo; Cross Reactions; Cytoskeleton; Dipodomys; Fluorescent Antibody Technique; Gelsolin; Isoelectric Point; Microfilament Proteins; Molecular Weight; Phosphofructokinase-1
PubMed: 3025844
DOI: 10.1073/pnas.83.24.9502 -
Biochemistry Feb 1991Six site-directed mutants of Escherichia coli phosphofructokinase (PFK) were made in an attempt to produce an enzyme "locked" in the inactive or "T"-state. The kinetic...
Six site-directed mutants of Escherichia coli phosphofructokinase (PFK) were made in an attempt to produce an enzyme "locked" in the inactive or "T"-state. The kinetic properties of the mutants were examined as a function of the substrates fructose 6-phosphate (Fru6P) and ATP, the positive effector GDP, and the negative effector phosphoenolpyruvate (PEP). All mutants exhibited lower activity than wild-type PFK. Three mutants (RS63, LV153, and VT246) had apparent dissociation constants for substrates and effectors similar to those of wild type. One mutant, HN160, had a 10-fold reduced affinity for Fru6P and reduced apparent affinity for the effectors. Two mutants, SN159 and T(GS)156, exhibited hyperbolic kinetics consistent with a "locked" T-state protein. Surprisingly, T(GS)156 showed hyperbolic activation in response to the physiological inhibitor PEP. The mutant PFK properties are discussed in terms of the PFK structure. These results suggest that the kinetic properties of PFK are sensitive to interactions in the homotropic interface; residues 156-160 in particular are critical in mediating the interactions between effector and active sites and in the T to R quaternary transition.
Topics: Adenosine Triphosphate; Allosteric Regulation; Base Sequence; Escherichia coli; Kinetics; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Oligonucleotide Probes; Phosphoenolpyruvate; Phosphofructokinase-1; Protein Conformation
PubMed: 1825177
DOI: 10.1021/bi00220a005 -
The Journal of Biological Chemistry Jul 1996In this work we used in vitro mutagenesis to modify the allosteric properties of the heterooctameric yeast phosphofructokinase. Specifically, we identified two amino... (Comparative Study)
Comparative Study
In this work we used in vitro mutagenesis to modify the allosteric properties of the heterooctameric yeast phosphofructokinase. Specifically, we identified two amino acids involved in the binding of the most potent allosteric activator fructose 2,6-bisphosphate. Thus, Ser724 was replaced by an aspartate and His859 by a serine in each of the enzyme subunits. Whereas the substitutions had no drastic effects when introduced only in one of the two types of subunits, kinetic parameters were modified when both subunits carried the mutation. Thus, the enzyme with His859 --> Ser showed an increase in Ka for binding of the activator, whereas the one with Ser724 --> Asp failed to react to the addition of fructose 2, 6-bisphosphate, at all. The enzymes still responded to other allosteric activators, such as AMP. Stabilities of the mutant subunits were not significantly altered in vivo, as judged from Western blot analysis. Phenotypically, strains expressing the mutant PFK genes showed a pronounced effect on the level of intermediary metabolites after growth on glucose. Mutants not responding to the activator at all (Ser724 --> Asp) also displayed higher generation times on glucose medium. This could be suppressed by increasing the gene dosage of the mutant alleles. These results indicate that fructose 2,6-bisphosphate through its activation of phosphofructokinase plays an important role in regulation of the glycolytic flux.
Topics: Allosteric Regulation; Allosteric Site; Amino Acid Sequence; Aspartic Acid; Base Sequence; DNA Primers; Enzyme Activation; Fructosediphosphates; Glycolysis; Histidine; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Oligodeoxyribonucleotides; Phosphofructokinase-1; Point Mutation; Polymerase Chain Reaction; Promoter Regions, Genetic; Recombinant Proteins; Saccharomyces cerevisiae; Serine
PubMed: 8663166
DOI: 10.1074/jbc.271.27.15928 -
Biochemistry Oct 1995Moderate concentrations of KSCN inactivate the allosteric phosphofructokinase from Escherichia coli by dissociating the subunit interface that contains the binding site...
Moderate concentrations of KSCN inactivate the allosteric phosphofructokinase from Escherichia coli by dissociating the subunit interface that contains the binding site for the substrate fructose-6-phosphate. At a given KSCN concentration, the activity varies with the concentration of protein as expected from a simple equilibrium between active tetramers and inactive dimers. The equilibrium constants for the dissociation of a tetramer into dimers have been determined in 0.4 M KSCN for the wild-type enzyme and the noncooperative mutant T125S, the hypercooperative mutant E148A-R152A, and the inactive mutant D127S. The stability of the tetrameric structure is decreased by the mutations E148A-R152A that are in the interface and increased by the mutation T125S that does not belong to it. There could be an inverse correlation between the cooperativity of the saturation by fructose-6-phosphate (in absence of any effector) and the stability of the interface that contains its binding site. Hybrid tetramers can be formed upon reassociation of a dimer from an active phosphofructokinase (wild-type, T125S, or E148-R152A) with a dimer from the inactive D127S mutant, and their stability and cooperativity toward fructose-6-phosphate have been measured without purifying them. The results indicate that the formation of a hybrid interface involves some flexibility of the two dimers and that the allosteric coupling between distant sites could be related to the plasticity and instability of the interactions across this interface.
Topics: Allosteric Regulation; Bacterial Proteins; Escherichia coli; Macromolecular Substances; Phosphofructokinase-1; Point Mutation; Protein Binding; Structure-Activity Relationship; Thiocyanates
PubMed: 7548084
DOI: 10.1021/bi00040a036 -
Biochemical and Biophysical Research... Apr 1971
Topics: Adenine Nucleotides; Adenosine Triphosphate; Drug Stability; Flavobacterium; Hexosephosphates; Hot Temperature; Kinetics; Phosphofructokinase-1; Pyruvates
PubMed: 4252961
DOI: 10.1016/s0006-291x(71)80081-5 -
Biochemistry Aug 1973
Topics: Adenosine Monophosphate; Adenosine Triphosphate; Ammonium Sulfate; Binding Sites; Chromatography, DEAE-Cellulose; Cyclic AMP; Enzyme Activation; Erythrocytes; Fructosephosphates; Hemolysis; Humans; Hydrogen-Ion Concentration; Kinetics; Light; Methylene Blue; Nucleotides; Oxidation-Reduction; Phosphofructokinase-1; Photochemistry; Structure-Activity Relationship
PubMed: 4354372
DOI: 10.1021/bi00742a024 -
European Journal of Biochemistry Apr 1972
Topics: Animals; Circular Dichroism; Fructosephosphates; Guanidines; Hydrogen-Ion Concentration; Macromolecular Substances; Mercaptoethanol; Molecular Weight; Muscles; Osmolar Concentration; Phosphofructokinase-1; Rabbits; Ultracentrifugation
PubMed: 4260953
DOI: 10.1111/j.1432-1033.1972.tb01785.x -
Journal of Theoretical Biology Jul 1983The kinetics of the phosphofructokinase reaction were studied by computer modeling. A general random order, two-state allosteric model, of which the...
The kinetics of the phosphofructokinase reaction were studied by computer modeling. A general random order, two-state allosteric model, of which the Monod--Wyman--Changeux model is a limiting case, was found to most accurately reproduce the experimental observations of Pettigrew & Frieden (1979 a,b). A simplified model with Hill coefficients was found to fit almost as well. In these models substrates bind preferentially to and stabilize the enzyme in the R state, and ATPH3-, the inhibitory species, binds preferentially to and stabilizes the enzyme in the T state. Enzymatic activity is regulated by conversion from the R to the T state, which is effected by protonation, especially of the uncomplexed enzyme, but the experimental data are inadequate for accurate estimation of the pKa of the enzyme. Random order binding of substrates is an important cause of sigmoidal kinetics. Additional experiments that would aid in the discrimination among rival models are described.
Topics: Adenosine Triphosphate; Computers; Kinetics; Models, Biological; Muscles; Phosphofructokinase-1
PubMed: 6225913
DOI: 10.1016/0022-5193(83)90030-9