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Biophysical Journal Apr 2021Although much is known about the biochemical regulation of glycolytic enzymes, less is understood about how they are organized inside cells. We systematically examine...
Although much is known about the biochemical regulation of glycolytic enzymes, less is understood about how they are organized inside cells. We systematically examine the dynamic subcellular localization of glycolytic protein phosphofructokinase-1/PFK-1.1 in Caenorhabditis elegans. We determine that endogenous PFK-1.1 localizes to subcellular compartments in vivo. In neurons, PFK-1.1 forms phase-separated condensates near synapses in response to energy stress from transient hypoxia. Restoring animals to normoxic conditions results in cytosolic dispersion of PFK-1.1. PFK-1.1 condensates exhibit liquid-like properties, including spheroid shapes due to surface tension, fluidity due to deformations, and fast internal molecular rearrangements. Heterologous self-association domain cryptochrome 2 promotes formation of PFK-1.1 condensates and recruitment of aldolase/ALDO-1. PFK-1.1 condensates do not correspond to stress granules and might represent novel metabolic subcompartments. Our studies indicate that glycolytic protein PFK-1.1 can dynamically form condensates in vivo.
Topics: Animals; Caenorhabditis elegans; Glycolysis; Organelles; Phosphofructokinase-1; Phosphofructokinases; Phosphorylation
PubMed: 32853565
DOI: 10.1016/j.bpj.2020.08.002 -
BMB Reports Nov 2023Most cancer cells utilize glucose at a high rate to produce energyand precursors for the biosynthesis of macromolecules such as lipids, proteins, and nucleic acids. This...
Most cancer cells utilize glucose at a high rate to produce energyand precursors for the biosynthesis of macromolecules such as lipids, proteins, and nucleic acids. This phenomenon is called the Warburg effect or aerobic glycolysis- this distinct characteristic is an attractive target for developing anticancer drugs. Here, we found that Phosphofructokinase-1 (PFK-1) is a substrate of the Protein Phosphatase 4 catalytic subunit (PP4C)/PP4 regulatory subunit 1 (PP4R1) complex by using immunoprecipitation and in vitro assay. While manipulation of PP4C/PP4R1 does not have a critical impact on PFK-1 expression, the absence of the PP4C/PP4R1 complex increases PFK-1 activity. Although PP4C depletion or overexpression does not cause a dramatic change in the overall glycolytic rate, PP4R1 depletion induces a considerable increase in both basal and compensatory glycolytic rates, as well as the oxygen consumption rate, indicating oxidative phosphorylation. Collectively, the PP4C/PP4R1 complex regulates PFK-1 activity by reversing its phosphorylation and is a promising candidate for treating glycolytic disorders and cancers. Targeting PP4R1 could be a more efficient and safer strategy to avoid pleiotropic effects than targeting PP4C directly. [BMB Reports 2023; 56(11): 618-623].
Topics: Phosphorylation; Phosphoprotein Phosphatases; Phosphofructokinase-1; Carbohydrate Metabolism; Glycolysis
PubMed: 37605615
DOI: 10.5483/BMBRep.2023-0065 -
Biochemistry Jan 2015The coupling between the binding of the substrate Fru-6-P and the inhibitor phospho(enol)pyruvate (PEP) in phosphofructokinase (PFK) from the extreme thermophile Thermus...
The coupling between the binding of the substrate Fru-6-P and the inhibitor phospho(enol)pyruvate (PEP) in phosphofructokinase (PFK) from the extreme thermophile Thermus thermophilus is much weaker than that seen in a PFK from Bacillus stearothermophilus. From the crystal structures of Bacillus stearothermophilus PFK (BsPFK) the residues at positions 59, 158, and 215 in BsPFK are located on the path leading from the allosteric site to the nearest active site and are part of the intricate hydrogen-bonding network connecting the two sites. Substituting the corresponding residues in Thermus thermophilus PFK (TtPFK) with the amino acids found at these positions in BsPFK allowed us to enhance the allosteric inhibition by PEP by nearly 3 kcal mol(-1) (50-fold) to a value greater than or equal to the coupling observed in BsPFK. Interestingly, each single variant N59D, A158T, and S215H produced a roughly 1 kcal mol(-1) increase in coupling free energy of inhibition. The effects of these variants were essentially additive in the three combinations of double variants N59D/A158T, N59D/S215H, and A158T/S215H as well as in the triple variant N59D/A158T/S215H. Consequently, while the hydrogen-bonding network identified is likely involved in the inhibitory allosteric communication, a model requiring a linked chain of interactions connecting the sites is not supported by these data. Despite the fact that the allosteric activator of the bacterial PFK, MgADP, binds at the same allosteric site, the substitutions at positions 59, 158, and 215 do not have an equally dramatic effect on the binding affinity and the allosteric activation by MgADP. The effect of the S215H and N59D/A158T/S215H substitutions on the activation by MgADP could not be determined because of a dramatic drop in MgADP binding affinity that resulted from the S215H substitution. The single variants N59D and A158T supported binding but showed little change in the free energy of activation by MgADP compared to the wild type TtPFK. These results support previous suggestions that heterotropic inhibition and activation occur by different pathways prokaryotic PFK.
Topics: Adenosine Diphosphate; Allosteric Regulation; Crystallography, X-Ray; Fructosephosphates; Geobacillus stearothermophilus; Hydrogen-Ion Concentration; Kinetics; Models, Molecular; Mutant Proteins; Phosphoenolpyruvate; Phosphofructokinase-1; Temperature; Thermus thermophilus
PubMed: 25531642
DOI: 10.1021/bi501127a -
Blood Aug 1979The subunit composition of phosphofructokinase from normal and malignant blood cells has been investigated by means of immunologic, electrophoretic, and chromatographic...
The subunit composition of phosphofructokinase from normal and malignant blood cells has been investigated by means of immunologic, electrophoretic, and chromatographic methods. Immunoprecipitation tests were performed with three specific antisera recognizing each of the basic subunits of human phosphofructokinase: muscle, M-type; liver, L-type; and fibroblast, F-type. Mature polymorphonuclear cells contain mainly L-subunits, while lymphocytes and platelets contain hybrids formed of L and F subunits; these hybrids can be electrophoretically separated. Red cell phosphofructokinase is composed of L and M subunits, as judged by its reactivity with anti-L and anti-M-type antisera. The various M-L hybrids composing red cell phosphofructokinase could be only separated by chromatography on DEAE-Cellulose. Lymphocytes from patients with chronic lymphocytic leukemia and lymphoblasts from patients with acute lymphoblastic leukemia contain phosphofructokinase forms similar to those from normal lymphocytes, while the immature granulocytic cells (leukemic myeloblasts and myeloid cells of chronic myeloid leukemia) are characterized by a reinforcement of enzyme inhibition by anti-F-type antiserum. Lymphoid lines in culture (Epstein-Barr virus (EBV)-induced or malignant lymphoma-derived lines) are characterized by the indistinctive expression of all three basic subunits, similar to that found in some fetal tissues. This article represents the first description of the isozymic nature of phosphofructokinase in platelets and white blood cells and of its changes with malignancy and cell culture. This enzyme might represent a useful marker in the characterization of the leukemic cells.
Topics: Antigens; Blood Cells; Blood Platelets; Erythrocytes; Granulocytes; Humans; Isoenzymes; Leukemia; Lymphocytes; Lymphoma; Neutrophils; Phosphofructokinase-1
PubMed: 156568
DOI: No ID Found -
FEBS Letters Jan 1996Muscle phosphofructokinase interacts with microtubule-associated protein-free microtubules resulting in a reduction of the overall activity of the enzyme [Lehotzky et...
Muscle phosphofructokinase interacts with microtubule-associated protein-free microtubules resulting in a reduction of the overall activity of the enzyme [Lehotzky et al. (1993) J. Biol. Chem. 268, 10888-10894] and periodical cross-linking of the tubules [Lehotzky et al. (1994) Biochem. Biophys. Res. Commun. 204, 585-591]. Microtubule polymers of 'tail-free' tubulin obtained by removal of the carboxy-termini with limited subtilisin digestion retain the binding domains for phosphofructokinase that cross-bridges microtubule 'bodies'. Microtubule-associated proteins bound on tubulin 'tails' do not perturb the kinase binding. These data suggest that the tubulin carboxy-terminal domain is not involved in microtubule-phosphofructokinase interactions and phosphofructokinase and microtubule-associated proteins have distinct binding domains on microtubules. Of different isoforms of phosphofructokinase, occurring mainly in brain and tumor cells, the muscle isoform exhibits selective adsorption behaviour on microtubules. Phosphofructokinase M and C isoforms with different associative and allosteric properties may represent an auxiliary pathway to modulate energy production via glycolysis.
Topics: Animals; Electrophoresis, Polyacrylamide Gel; Isoenzymes; Kinetics; Microscopy, Electron; Microtubule-Associated Proteins; Microtubules; Muscle, Skeletal; Phosphofructokinase-1; Protein Binding; Rabbits; Subtilisins
PubMed: 8635590
DOI: 10.1016/0014-5793(95)01510-8 -
Proceedings of the National Academy of... May 1981We present an analysis of glycolysis based on experimental findings and an interpretation based on concepts of efficiency, resonance response, and control features...
We present an analysis of glycolysis based on experimental findings and an interpretation based on concepts of efficiency, resonance response, and control features available in highly nonlinear reaction kinetics. We begin with a model for the glycolytic mechanism that is comprehensive, includes a large number of known activations and inhibitions of enzymes by metabolites, and couples the phosphofructokinase (PFKase) and the pyruvate kinase (PKase) reactions. The PFKase and PKase reactions and the coupling between them are modeled according to experimental information, but we do not attempt to model the glyceraldehyde-3-phosphate dehydrogenase-3-phosphoglycerate kinase reaction. We use experimental data to obtain the best estimates for the kinetic parameters and test the model by calculating the concentration variations of the intermediate metabolites. We confirm oscillatory behavior and calculate the ATP/ADP ratio and the free-energy dissipation for an extended range of the kinetic parameters as a function of the driving force for the glycolytic pathway, a measure of which is the total adenine nucleotide concentration. We find agreement of the calculated results with experimental findings except for the insufficiently represented reactions. Our model shows that the average ATP/ADP ratio is increased and the average free-energy dissipation is decreased in an oscillatory compared with a steady state mode of operation. The average values of the ATP/ADP ratio and of the free energy dissipation change abruptly past the onset of sustained oscillations.
Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Glycolysis; Kinetics; Mathematics; Models, Biological; Phosphofructokinase-1; Pyruvate Kinase
PubMed: 6454892
DOI: 10.1073/pnas.78.5.2952 -
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 Biochemical Journal Nov 19711. The properties of phosphofructokinase after its slight purification from the mucosa of rat jejunum were studied. 2. The enzyme is inhibited by almost 100% by an...
1. The properties of phosphofructokinase after its slight purification from the mucosa of rat jejunum were studied. 2. The enzyme is inhibited by almost 100% by an excess of ATP (1.6mm), with 0.2mm-fructose 6-phosphate. AMP, ADP, P(i) and NH(4) (+) at 0.2, 0.76, 1.0 and 2mm respectively do not individually prevent the inhibition of phosphofructokinase activity by 1.6mm-ATP with 0.2mm-fructose 6-phosphate to any great extent, but all of them together completely prevent the inhibition of phosphofructokinase by ATP. 3. One of the effects of high concentrations of ATP on the enzyme was to increase enormously the apparent K(m) value for the other substrate fructose 6-phosphate, and this increase is largely counteracted by the presence of AMP, ADP, P(i) and NH(4) (+). At low concentrations of ATP the above effectors individually decrease the concentration of fructose 6-phosphate required for half-maximum velocity and when present together they decrease it further, in a more than additive way. 4. When fructose 6-phosphate is present at a saturating concentration (5mm), 0.3mm-NH(4) (+) increases the maximum velocity of the reaction 3.3-fold; with 0.5mm-fructose 6-phosphate, 4.5mm-NH(4) (+) is required for maximum effect. The other effectors do not change the maximum reaction velocity. 5. The results presented here suggest that NH(4) (+), AMP, ADP and P(i) synergistically decrease the inhibition of phosphofructokinase activity at high concentrations of ATP by decreasing the concentration of fructose 6-phosphate required for half-maximum velocity. Such synergism among the effectors and an observed, low ;energy charge' [(ATP+(1/2)ADP)/(AMP+ADP+ATP)] in conjunction with the possibility of a relatively high NH(4) (+) and fructose 6-phosphate concentration in this tissue, may keep the mucosal phosphofructokinase active and uninhibited by ATP under aerobic conditions, thus explaining the high rate of aerobic glycolysis and the lack of Pasteur effect in this tissue.
Topics: Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Aerobiosis; Animals; Female; Fructosephosphates; Glycolysis; Intestinal Mucosa; Jejunum; Kinetics; Oxygen; Phosphates; Phosphofructokinase-1; Quaternary Ammonium Compounds; Rats
PubMed: 4259410
DOI: 10.1042/bj1250507 -
Applied and Environmental Microbiology May 2015Escherichia coli that is unable to metabolize d-glucose (with knockouts in ptsG, manZ, and glk) accumulates a small amount of d-glucose (yield of about 0.01 g/g) during...
Escherichia coli that is unable to metabolize d-glucose (with knockouts in ptsG, manZ, and glk) accumulates a small amount of d-glucose (yield of about 0.01 g/g) during growth on the pentoses d-xylose or l-arabinose as a sole carbon source. Additional knockouts in the zwf and pfkA genes, encoding, respectively, d-glucose-6-phosphate 1-dehydrogenase and 6-phosphofructokinase I (E. coli MEC143), increased accumulation to greater than 1 g/liter d-glucose and 100 mg/liter d-mannose from 5 g/liter d-xylose or l-arabinose. Knockouts of other genes associated with interconversions of d-glucose-phosphates demonstrate that d-glucose is formed primarily by the dephosphorylation of d-glucose-6-phosphate. Under controlled batch conditions with 20 g/liter d-xylose, MEC143 generated 4.4 g/liter d-glucose and 0.6 g/liter d-mannose. The results establish a direct link between pentoses and hexoses and provide a novel strategy to increase carbon backbone length from five to six carbons by directing flux through the pentose phosphate pathway.
Topics: Arabinose; Escherichia coli; Escherichia coli Proteins; Fermentation; Glucose; Glucosephosphate Dehydrogenase; Mannose; Metabolic Engineering; Pentose Phosphate Pathway; Phosphofructokinase-1; Xylose
PubMed: 25746993
DOI: 10.1128/AEM.04058-14 -
The Journal of Clinical Investigation Jun 1993In patients with non-insulin-dependent diabetes mellitus (NIDDM) and matched control subjects we examined the interrelationships between in vivo nonoxidative glucose... (Comparative Study)
Comparative Study
In patients with non-insulin-dependent diabetes mellitus (NIDDM) and matched control subjects we examined the interrelationships between in vivo nonoxidative glucose metabolism and glucose oxidation and the muscle activities, as well as the immunoreactive protein and mRNA levels of the rate-limiting enzymes in glycogen synthesis and glycolysis, glycogen synthase (GS) and phosphofructokinase (PFK), respectively. Analysis of biopsies of quadriceps muscle from 19 NIDDM patients and 19 control subjects showed in the basal state a 30% decrease (P < 0.005) in total GS activity and a 38% decrease (P < 0.001) in GS mRNA/microgram DNA in NIDDM patients, whereas the GS protein level was normal. The enzymatic activity and protein and mRNA levels of PFK were all normal in diabetic patients. In subgroups of NIDDM patients and control subjects an insulin-glucose clamp in combination with indirect calorimetry was performed. The rate of insulin-stimulated nonoxidative glucose metabolism was decreased by 47% (P < 0.005) in NIDDM patients, whereas the glucose oxidation rate was normal. The PFK activity, protein level, and mRNA/microgram DNA remained unchanged. The relative activation of GS by glucose-6-phosphate was 33% lower (P < 0.02), whereas GS mRNA/micrograms DNA was 37% lower (P < 0.05) in the diabetic patients after 4 h of hyperinsulinemia. Total GS immunoreactive mass remained normal. In conclusion, qualitative but not quantitative posttranslational abnormalities of the GS protein in muscle determine the reduced insulin-stimulated nonoxidative glucose metabolism in NIDDM.
Topics: Adult; Aerobiosis; Aged; Anaerobiosis; Diabetes Mellitus, Type 2; Female; Glucose; Glucose Clamp Technique; Glycogen Synthase; Humans; Insulin Resistance; Lipid Metabolism; Male; Metabolic Clearance Rate; Middle Aged; Muscles; Oxidation-Reduction; Phosphofructokinase-1; RNA, Messenger; White People
PubMed: 8514849
DOI: 10.1172/JCI116466