Fructose 6-phosphate 1-phosphotransferase Research Articles

Measurement of the inorganic pyrophosphate in tissues of Pisum sativum L.

Purified pyrophosphate: fructose 6-phosphate 1-phosphotransferase (EC 2.7.1.90) was used to measure the inorganic pyrophosphate in unfractionated extracts of tissues of Pisum sativum L. The fructose 1,6-bisphosphate produced by the above enzyme was measured by coupling to NADH oxidation via aldolase (EC 4.1.2.13), triosephosphate isomerase (EC 5.3.1.1) and glycerol-3-phosphate dehydrogenase (EC 1.1.1.8). Amounts of pyrophosphate as low as 1 nmol could be measured. The contents of pyrophosphate in the developing embryo of pea, and in the apical 2 cm of the roots, were appreciable; 9.4 and 8.9 nmol g(-1) fresh weight, respectively. The possibility that pyrophosphate acts in vivo as an energy source for pyrophosphate: fructose 6-phosphate 1-phosphotransferase and for UDPglucose pyrophosphorylase (EC 2.7.7.9) is considered.

Inhibition by Metabolic Intermediates of Pyrophosphate : Fructose 6-Phosphate Phosphotransferase from Germinating Castor Bean Endosperm

Summary The aim of this work was to study the effects of metabolic intermediates on pyrophosphate: fructose 6-phosphate phosphotransferase (PFP) from endosperm of germinating castor bean ( Ricinus communis ). Glucose 1-phosphate, glucose 6-phosphate, UDPglucose, glucose, fructose, sucrose and pyruvate, each at 1 mM, and malate, succinate and glycolate, each at up to 5mM, had no significant effect on enzyme activity. PFP was inhibited by 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, citrate and all nucleotides tested. Inhibition by MgATP, 3-phosphoglycerate, phosphoenolpyruvate and citrate was due primarily to their effect on the affinity of PFP for fructose 2,6-bisphosphate, a potent activator of the enzyme. These inhibitors each increased the concentration of fructose 2,6-bisphosphate required for half maximum activation of PFP up to 30-fold, from 6 to 180 nanomolar. The possible roles of these compounds in the control of PFP are discussed.

Fructose 2,6-bisphosphate as a contaminant of commercially obtained fructose 6-phosphate: Effect on PP i:fructose 6-phosphate phosphotransferase

Fructose 6-phosphate from several commercial sources was shown to be contaminated with fructose 2,6-bisphosphate. This contaminant was identified by its activation of PP i:fructose 6-phosphate phosphotransferase, extreme acid lability and behaviour on ion-exchange chromatography. The apparent kinetic properties of PP i:fructose 6-phosphate phosphotransferase from castor bean endosperm were considerably altered when contaminated fructose 6-phosphate was used as a substrate. Varying levels of fructose 2,6-bisphosphate in the substrate may account for differences that have been observed in the properties of the above enzyme from several plant sources.

The control of glycolysis and gluconeogenesis by protein phosphorylation.

Fructose 2,6-bisphosphate has been discovered as a potent stimulator of liver phosphofructokinase. It is also an inhibitor of fructose 1,6-biphosphatase and a stimulator of PPi: fructose 6-phosphate phosphotransferase from higher plants. It is formed from fructose 6-phosphate and ATP by a 6-phosphofructo 2-kinase and hydrolysed by a fructose 2,6-bisphosphatase. These two enzymes have very similar physicochemical properties and could not be separated from each other. They are substrates for cyclic-AMP-dependent protein kinase, which inactivates the first enzyme and activates the second.

Pyrophosphate: fructose 6-phosphate phosphotransferase in germinating castor bean seedlings

The distribution of enzymes interconverting fructose 6-phosphate and fructose 1,6-bisphosphate has been studied in a range of tissues from castor bean seedlings. In each tissue the activity of PP i:fructose 6-phosphate phosphotransferase was greater than phosphofructokinase and substantial compared with fructose 1,6-bisphosphatase. PP i:fructose 6-phosphate phosphotransferase in endosperm is apparently confined to the cytoplasm. The role of this latter enzyme in vivo is discussed.

D-Fructose 2,6-bisphosphate: A naturally occurring activator for inorganic pyrophosphate:D-fructose-6-phosphate 1-phosphotransferase in plants

Inorganic pyrophosphate:D-fructose-6-phosphate 1-phosphotransferase from mung beans ( Phaseolus aureus Roxb. ) was activated markedly by D-fructose 2,6-bisphosphate, with a K A of about 50 nM. The enzyme exhibited hyperbolic kinetics both in the absence and presence of the activator. D-Fructose 2,6-bisphosphate (1 μM) decreased the K m for D-fructose 6-phosphate 67-fold (from 20 mM to 0.3 mM) and increased the V max 15-fold; these two effects combined to give a 500-fold activation at 0.3 mM D-fructose 6-phosphate. In contrast, ATP:D-fructose 6-phosphate 1-phosphotransferase from the same source was found not to be affected by D-fructose 2,6-bisphosphate. A natural activator for inorganic pyrophosphate:D-fructose 6-phosphate 1-phosphotransferase was isolated from mung-bean extracts and identified as D-fructose 2,6-bisphosphate.

Permeabilization of animal cells for kinetic studies of intracellular enzymes: in situ behavior of the glycolytic enzymes of erythrocytes.

Intracellular enzymes in erythrocytes can be made accessible for in situ kinetic studies by treating the cells with bifunctional reagents to crosslink proteins, thus creating a network that allows subsequent permeabilization by delipidation without escape of intracellular proteins. Dimethyl suberimidate, dimethyl 3,3'-dithiobispropionimidate, and toluene-2,4-diisocyanate have been used successfully as crosslinking reagents, and digitonin has been used for delipidation. In a systematic study of the in situ behavior of the 11 glycolytic enzymes of rat erythrocytes, it was observed that Km and Vmax values for the majority of the enzymes are essentially the same in situ as in vitro. Lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) is inhibited by excess of pyruvate as much in situ as in vitro. Hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) was allosterically inhibited by glucose 6-phosphate nearly as much in situ as in vitro but was not affected by 2,3-biphosphoglycerate. The allosteric properties of 6-phosphofructokinase (ATP:D-fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11), glyceraldehyde-phosphate dehydrogenase [D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12], and pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) in situ were qualitatively similar to those observed in vitro, but some important quantitative differences were noticed. Particularly striking was the much greater activity of phosphofructokinase in situ compared to that in vitro at physiological concentrations of effector metabolites.

Inhibition of phosphofructokinase by the toxic cembranolide sarcophine isolated from the soft-bodied coral Sarcophyton glaucum

Sarcophine, a toxic cembranolide extracted from Sarcophyton glaucum, is known to have toxic effects on fishes, mice and rats, and also reacts with cysteine. Evidence is herewith presented to show that sarcophine inhibits the enzyme phosphofructokinase (ATP: d fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11) purified from rabbit skeletal muscle. Linear uncompetitive inhibition of the enzyme is indicated due to the reaction of the sarcophine with thiol groups of the enzyme. Sarcophine is shown to be a reversible inhibitor, and a model of the reaction course is offered.

Studies on the Reaction Mechanism of Skeletal Muscle Phosphofructokinase

Abstract The kinetics and mechanism of action of rabbit skeletal muscle phosphofructokinase (ATP:d-fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11) were investigated by several techniques. The initial velocity, product inhibition, isotope exchange patterns, and the result of a pulse labeling experiment are consistent with a mechanism in which ADP (or inosine diphosphate) dissociates from the enzyme before the addition of fructose 6-phosphate, and suggest the formation of phosphoryl enzyme as an intermediate. The enzyme preparation shows ATPase and fructose diphosphatase activities. The ATPase activity is catalyzed by the same protein that catalyzes the phosphofructokinase reaction.