-
Archives of Biochemistry and Biophysics May 1998The phosphorylation state of phosphofructokinase from the mantle tissue of the facultative anaerobe mollusk Mytilus galloprovincialis was determined by a...
The phosphorylation state of phosphofructokinase from the mantle tissue of the facultative anaerobe mollusk Mytilus galloprovincialis was determined by a back-phosphorylation technique. The incubation of intact mantle tissue with 8-bromoadenosine 3':5'-cyclic monophosphate increased significantly the phosphate content of phosphofructokinase, which indicates that the enzyme can be phosphorylated in vivo by endogenous cAMP-dependent protein kinase. The phosphate content of mussel phosphofructokinase changes significantly during the year, in agreement with the kinetic data that show a more active enzyme form in earlier autumn. These results suggest that cAMP-dependent phosphorylation of phosphofructokinase can be partially responsible for the observed glycolytic changes associated with the annual gametogenic cycle that takes place in the mantle tissue of the mollusk. On the contrary, no differences were observed between aerobic and 24-h hypoxic mussels with regard to the phosphorylation state and the kinetic constants of phosphofructokinase. This result is inconsistent with the hypothesis that phosphorylation of phosphofructokinase is involved in the glycolytic depression that takes place during the long-term environmental hypoxia that the mollusk can undergo.
Topics: Animals; Bivalvia; Cyclic AMP-Dependent Protein Kinases; Glycolysis; Kinetics; Organophosphates; Oxygen; Phosphofructokinase-1; Phosphorylation; Seasons
PubMed: 9606959
DOI: 10.1006/abbi.1998.0631 -
European Journal of Biochemistry Aug 1982Escherichia coli contains a major phosphofructokinase isoenzyme, phosphofructokinase 1, which is allosteric, and a minor isoenzyme, phosphofructokinase 2. The pfkB1...
Escherichia coli contains a major phosphofructokinase isoenzyme, phosphofructokinase 1, which is allosteric, and a minor isoenzyme, phosphofructokinase 2. The pfkB1 mutation is known to increase the amount of phosphofructokinase 2 and allow growth on sugars of mutants lacking phosphofructokinase 1; it does not affect growth on substances such as glycerol or lactate (i.e., 'gluconeogenic growth'). However, gluconeogenic growth is markedly impaired in strains with a different allele, pfkB1*. We show here that strains with pfkB1* contain an altered form of phosphofructokinase 2, called phosphofructokinase 2*, which has been purified. Phosphofructokinase 2* is cold labile and has slightly different kinetic characteristics from phosphofructokinase 2, which include being less sensitive to inhibition by fructose 1,6-bisphosphate. The Km for fructose 6-phosphate is low (about 5 X 10(-5) M) in both phosphofructokinase 2 and phosphofructokinase 2*. However, in strains lacking phosphofructokinase 1, a high level of phosphofructokinase 2 is associated with unusually high concentrations of hexose monophosphates during growth on glucose, while a strain with phosphofructokinase 2* instead of phosphofructokinase 2 grows more rapidly on glucose and contains lower levels of hexose monophosphates. In gluconeogenic conditions, by contrast, hexose monophosphate levels are normal in phosphofructokinase 2 strains, while the impaired growth of phosphofructokinase 2* strains is associated with high levels of fructose 2,6-bisphosphate and very low levels of hexose monophosphates. These results show that phosphofructokinase 2, as studied in vitro, should no longer be regarded as a 'non-allosteric' protein, a conclusion also reached by Kotlarz and Buc on the basis of different types of experiments [Eur. J. Biochem. 117, 569-574 (1981)]. The fact that mutational alteration of phosphofructokinase 2 allows more rapid growth on glucose but severely impairs gluconeogenic growth is an indication of the significance of the regulation in vivo. The more rapid growth of the mutant on glucose might be explained on the basis of decreased sensitivity to an inhibitor (possibly, but not necessarily, fructose 1,6-bisphosphate), although other models are possible. A variety of speculations are offered as to the mechanism of gluconeogenic impairment.
Topics: Carbohydrates; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Gluconeogenesis; Isoenzymes; Phosphofructokinase-1
PubMed: 6215246
DOI: 10.1111/j.1432-1033.1982.tb06790.x -
Biochemistry Jan 2014An investigation into the kinetics and regulatory properties of the type-1 phosphofructokinase (PFK) from the extreme thermophile Thermus thermophilus (TtPFK) reveals an...
An investigation into the kinetics and regulatory properties of the type-1 phosphofructokinase (PFK) from the extreme thermophile Thermus thermophilus (TtPFK) reveals an enzyme that is inhibited by PEP and activated by ADP by modifying the affinity exhibited for the substrate fructose 6-phosphate (Fru-6-P) in a manner analogous to other prokaryotic PFKs. However, TtPFK binds both of these allosteric ligands significantly more tightly than other bacterial PFKs while effecting a substantially more modest extent of inhibition or activation at 25 °C, reinforcing the principle that binding affinity and effectiveness can be both independent and uncorrelated to one another. These properties have allowed us to establish rigorously that PEP only inhibits by antagonizing the binding of Fru-6-P and not by influencing turnover, a conclusion that requires kcat to be determined under conditions in which both inhibitor and substrate are saturating simultaneously. In addition, the temperature dependence of the allosteric effects on Fru-6-P binding indicate that the coupling free energies are entropy-dominated, as observed previously for PFK from Bacillus stearothermophilus but not for PFK from Escherichia coli , supporting the hypothesis that entropy-dominated allosteric effects may be a characteristic of enzymes derived from thermostable organisms. For such enzymes, the root cause of the allosteric effect may not be easily discerned from static structural information such as that obtained from X-ray crystallography.
Topics: Adenosine Diphosphate; Allosteric Regulation; Entropy; Fructosephosphates; Kinetics; Ligands; Phosphoenolpyruvate; Phosphofructokinase-1; Temperature; Thermus thermophilus
PubMed: 24328040
DOI: 10.1021/bi401402j -
Biochemistry Jul 2018Most reported bacterial phosphofructokinases (Pfks) are tetramers that exhibit activity allosterically regulated via conformational changes between the R and T states....
Most reported bacterial phosphofructokinases (Pfks) are tetramers that exhibit activity allosterically regulated via conformational changes between the R and T states. We report that the Pfk from Staphylococcus aureus NCTC 8325 ( SaPfk) exists as both an active tetramer and an inactive dimer in solution. Multiple effectors, including pH, ADP, ATP, and adenylyl-imidodiphosphate (AMP-PNP), cause equilibrium shifts from the tetramer to dimer, whereas the substrate F6P stabilizes SaPfk tetrameric assembly. Crystal structures of SaPfk in complex with different ligands and biochemical analysis reveal that the flexibility of the Gly150-Leu151 motif in helix α7 plays a role in tetramer-dimer conversion. Thus, we propose a molecular mechanism for allosteric regulation of bacterial Pfk via conversion between the tetramer and dimer in addition to the well-characterized R-state/T-state mechanism.
Topics: Allosteric Regulation; Crystallography, X-Ray; Humans; Models, Molecular; Phosphofructokinase-1; Protein Conformation; Protein Multimerization; Staphylococcal Infections; Staphylococcus aureus; Substrate Specificity
PubMed: 29940104
DOI: 10.1021/acs.biochem.8b00028 -
Archives of Biochemistry and Biophysics Dec 1984Phosphofructokinase (PFK) was purified from foot muscle of aerobic and anaerobic (24 h of anoxia) whelks, Busycotypus canaliculatum. Fructose-6-P kinetics were sigmoidal...
Phosphofructokinase (PFK) was purified from foot muscle of aerobic and anaerobic (24 h of anoxia) whelks, Busycotypus canaliculatum. Fructose-6-P kinetics were sigmoidal at pH 7.0 with affinity constants, S0.5, of 2.18 +/- 0.10 (nH = 2.5 +/- 0.1) and 2.48 +/- 0.13 mM (nH = 2.7 +/- 0.1) for the enzyme from aerobic verus anaerobic muscle. Affinity for ATP, like that for fructose-6-P, did not differ for the two enzymes (0.031 +/- 0.003 for the aerobic vs 0.041 +/- 0.007 mM for the anaerobic enzyme), but S0.5 for Mg2+ was significantly different for the two enzymes (0.060 +/- 0.006 vs 0.130 +/- 0.020 mM). Whelk muscle PFK was activated by NH+4, Pi, AMP, ADP, and fructose-2,6-P2.NH+4 and fructose-2,6-P2 were less effective activators of PFK from anoxic muscle, with apparent Ka's 1.6- and 3.5-fold higher for the anaerobic vs aerobic enzyme. Activators decreased S0.5 for fructose-6-P and reduced nH. With the exception of fructose-2,6-P2, the effects of activators on S0.5 were the same for the enzyme from aerobic and anaerobic muscle; fructose-2,6-P2 at 2.5 microM reduced S0.5 by only 3.3-fold for the anaerobic enzyme compared to 5.5-fold for the aerobic enzyme. ATP was a strong substrate inhibitor of PFK; the enzyme from anaerobic muscle showed greater ATP inhibition, with I50's 1.5- to 2.0-fold lower than those for the aerobic enzyme. The kinetic differences between PFK from anaerobic versus aerobic foot muscle (stronger ATP inhibition and decreased sensitivity to activators for the anaerobic enzyme) were consistent with kinetic differences reported for the phosphorylated versus dephosphorylated forms, respectively, of PFK in other systems. Treatment of PFK from anaerobic muscle with alkaline phosphatase resulted in a decrease in the Ka for fructose-2,6-P2 to a level similar to that of the aerobic enzyme. The physiological stress of anoxia may, therefore, induce a covalent modification of PFK.
Topics: Adenosine Triphosphate; Anaerobiosis; Animals; Chemical Phenomena; Chemistry; Enzyme Activation; Fructosephosphates; Kinetics; Muscles; Phosphofructokinase-1; Phosphorylation; Snails; Substrate Specificity
PubMed: 6240229
DOI: 10.1016/0003-9861(84)90242-x -
Methods in Enzymology 1982
Topics: Entamoeba histolytica; Kinetics; Molecular Weight; NAD; Phosphofructokinase-1; Spectrophotometry, Ultraviolet
PubMed: 6296625
DOI: 10.1016/s0076-6879(82)90113-6 -
Biochemical and Biophysical Research... Mar 1990Stopped-flow measurements have been carried out to study some basic allosteric properties of muscle and yeast phosphofructokinase at physiological concentration of...
Stopped-flow measurements have been carried out to study some basic allosteric properties of muscle and yeast phosphofructokinase at physiological concentration of enzyme. An important increase in the affinity for fructose-6-P accompanied by an intense decrease in the ATP inhibition was observed with the muscle enzyme, which also became insensitive to fructose-2,6-P2 under these conditions. Yeast phosphofructokinase exhibited a significant diminution in the inhibition by ATP, although with no apparent change in the affinity for fructose-6-P. These results provide strong support in favor of the dependence of the allosteric regulation of phosphofructokinase on its concentration in vivo.
Topics: Adenosine Triphosphate; Animals; Fructosediphosphates; Homeostasis; Kinetics; Muscles; Phosphofructokinase-1; Rabbits
PubMed: 2138893
DOI: 10.1016/0006-291x(90)90653-5 -
Comparative Biochemistry and... 19891. Phosphofructokinase from porcine heart, liver and erythrocytes were purified by affinity chromatographies on Cibacron Blue Sepharose and N6-ATP agarose. 2.... (Comparative Study)
Comparative Study
1. Phosphofructokinase from porcine heart, liver and erythrocytes were purified by affinity chromatographies on Cibacron Blue Sepharose and N6-ATP agarose. 2. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the heart and liver enzymes consist of only one kind of subunit, namely, M and L type subunits, respectively, whereas the erythrocyte enzyme comprises of three kinds of subunits, M, L and C types. 3. Some kinetic and regulatory properties of the enzymes were also measured.
Topics: Animals; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Isoenzymes; Kinetics; Liver; Myocardium; Phosphofructokinase-1; Swine
PubMed: 2527141
DOI: 10.1016/0305-0491(89)90369-6 -
Journal of the Royal Society, Interface Feb 2024In many living organisms displaying circadian rhythms, the intake of energy often occurs in a periodic manner. Glycolysis is a prototypical biochemical reaction that...
In many living organisms displaying circadian rhythms, the intake of energy often occurs in a periodic manner. Glycolysis is a prototypical biochemical reaction that exhibits a self-sustained oscillation under continuous injection of glucose. Here we study the effect of periodic injection of glucose on the glycolytic oscillation from a dynamical systems perspective. In particular, we employ Goldbeter's allosteric model of phosphofructokinase as a model system for glycolytic oscillations, and explore the effect of periodic substrate influx of varying frequencies and amplitudes by building the phase diagrams of Lyapunov exponents and oscillatory periods. When the frequency of driving is tuned around the harmonic and sub/super-harmonic conditions of the natural frequency, the system is entrained to a frequency-locked state, forming an entrainment band that broadens with an increasing amplitude of driving. On the other hand, if the amplitude is substantial, the system may transition, albeit infrequent, to a chaotic state which defies prediction of dynamical behaviour. Our study offers in-depth understandings into the controllability of glycolytic oscillation as well as explaining physical underpinnings that enable the synchronous oscillations among a dense population of cells.
Topics: Models, Biological; Circadian Rhythm; Phosphofructokinase-1; Glycolysis; Glucose
PubMed: 38350614
DOI: 10.1098/rsif.2023.0588 -
The Journal of Biological Chemistry Feb 1983Mutants of Saccharomyces cerevisiae completely lacking the soluble glycolytic enzyme fructose-6-P kinase are described. The mutations are semidominant, do not complement...
Mutants of Saccharomyces cerevisiae completely lacking the soluble glycolytic enzyme fructose-6-P kinase are described. The mutations are semidominant, do not complement one another, and define a gene PFK1 located 28-cm distal to rna1 on the extended right arm of chromosome XIII. Of 10 independent mutants, 3 can be suppressed by ochre suppressors. All mutants examined synthesize proteins that cross-react to the antibody against the purified yeast P-fructokinase. The enzyme in spontaneous revertants is distinguishable from the wild type enzyme with respect to thermolability and ATP inhibition. The locus PFK1 thus defines the structural gene of the enzyme. The pfk1 mutants are not leaky in vivo. All the glucose consumed by a double mutant lacking both P-fructokinase and 6-P-gluconate dehydrogenase ends up as 6-P-gluconate, yet the pfk1 mutants can glycolyze and grow on glucose in air. The cell mass produced per unit of glucose also remains unchanged. Anaerobically, however, growth does not take place, nor does glycolysis. P-fructokinase is thus a dispensable enzyme for aerobic growth, but indispensable for anaerobic growth. The properties of pfk1 mutants suggest that yeast has an alternative mechanism for the aerobic metabolism of fructose-6-P, presumably through the recently reported particulate P-fructokinase (Lobo, Z., and Maitra, P. K. (1982) FEBS Lett. 137, 279-282).
Topics: Aerobiosis; Anaerobiosis; Diploidy; Genetic Complementation Test; Glycolysis; Haploidy; Kinetics; Mutation; Phosphofructokinase-1; Saccharomyces cerevisiae
PubMed: 6218165
DOI: No ID Found