Product inhibition of potato tuber pyrophosphate:fructose-6-phosphate phosphotransferase by phosphate and pyrophosphate

The product inhibition of potato (Solanum tuberosum) tuber pyrophosphate:fructose-6-phosphate phosphotransferase by inorganic pyrophosphate and inorganic phosphate has been studied. The binding of substrates for the forward (glycolytic) and the reverse (gluconeogenic) reaction is random order, and occurs with only weak competition between the substrate pair fructose-6-phosphate and pyrophosphate, and between the substrate pair fructose-1,6-bisphosphate and phosphate. Pyrophosphate is a powerful inhibitor of the reverse reaction, acting competitively to fructose-1,6-biphosphate and noncompetitively to phosphate. At the concentrations needed for catalysis of the reverse reaction, phosphate inhibits the forward reaction in a largely noncompetitive mode with respect to both fructose-6-phosphate and pyrophosphate. At higher concentrations, phosphate inhibits both the forward and the reverse reaction by decreasing the affinity for fructose-2,6-bisphosphate and thus, for the other three substrates. These results allow a model to be proposed, which describes the interactions between the substrates at the catalytic site. They also suggest the enzyme may be regulated in vivo by changes of the relation between metabolites and phosphate and could act as a means of controlling the cytosolic pyrophosphate concentration.     

Wound-induced respiration and pyrophosphate:fructose-6-phosphate phosphotransferase in potato tubers

A seven fold increase in the rate of respiratory O2 uptake was observed 24 h after slicing of potato tuber disks. The maximum activity of pyrophosphate:fructose-6-phosphate phosphotransferase (PFP) was 5-7 times greater than that of ATP-dependent phosphofructokinase (PFK) in fresh or aged potato slices. Thus, PFP may participate in glycolysis which supplies respiratory substrate in potato tubers. The PFP activity of desalted extracts determined in the absence of fructose-2,6-bisphosphate (F2,6BP) increased by 4.5 fold 24 h after slicing. However, maximal PFP activity determined with saturating (1 microM) F2,6BP was not changed. The Ka values of PFP for F2,6BP was lowered from 33 to 7 nM after 24 h of aging treatment. This increased susceptibility of the PFP activity to its allosteric activator, F2,6BP, may be involved in the increased respiration in wounded disks of potato tubers. Immunoblotting experiments indicated that both the alpha (66 kDa) and the beta (60 kDa) subunits of PFP were present in fresh or 24 h aged tuber slices.     

High-yield purification of potato tuber pyrophosphate: fructose-6-phosphate 1-phosphotransferase.

The procedure of Yuan et al. (1988, Biochem. Biophys. Res. Commun. 154, 111-117) for the isolation of potato pyrophosphate:fructose-6-phosphate 1-phosphotransferase (PFP) has been modified so that a high yield of homogeneous enzyme could be obtained. Modifications included a lower temperature heat step, a lower percentage initial polyethylene glycol fractionation step (0 to 4%, w/v), stepwise elution following an increase from 30 to 50 mM pyrophosphate during affinity chromatography on Whatman P11 phosphocellulose, anion-exchange chromatography using Q-Sepharose "Fast Flow," and gel filtration chromatography with Superose 6 "Prep grade." Our procedure resulted in an overall 42% yield and a final specific activity of 87 mumol fructose 1,6-bisphosphate produced per minute per milligram protein. Rabbit anti-(potato PFP) polyclonal antibodies effectively immunoprecipitated the activity of both the pure enzyme and the enzyme from a crude extract. Western blot analysis demonstrated that the antibodies were monospecific for PFP. A survey of various potato cultivars demonstrated significant differences in PFP activity with respect to fresh weight. This observation should be taken into consideration before any purification of potato PFP is undertaken.     

Kinetic properties of pyrophosphate:fructose-6-phosphate phosphotransferase from germinating castor bean endosperm

Pyrophosphate:fructose-6-phosphate phosphotransferase (PFP) was purified over 500-cold from endosperm of germinating castor bean (Ricinus commiunis L. var. Hale). The kinetic properties of the purified enzyme were studied. PFP was specific for pyrophosphate and had a requirement for a divalent metal ion. The pH optimum for activity was 7.3 to 7.7. The enzyme had similar activities in the forward and reverse directions and exhibited hyperbolic kinetics with all substrates. Kinetic constants were determined in the presence of fructose 2,6-bisphosphate, which stimulated activity about 20-fold and increased the affinity of the enzyme for fructose 6-phosphate, fructose 1,6-bisphosphate, and pyrophosphate up to 10-fold. Half-maximum activation of PFP by fructose 2,6-bisphosphate was obtained at 10 nanomolar. The affinity of PFP for this activator was reduced by decreasing the concentration of fructose 6-phosphate or increasing that of phosphate. Phosphate inhibited PFP when the reaction was measured in the reverse direction, i.e. fructose 6-phosphate production. In the presence of fructose 2,6-bisphosphate, phosphate was a mixed inhibitor with respect to both fructose 6-phosphate and pyrophosphate when the reaction was measured in the forward direction, i.e. fructose 1,6-bisphosphate production. The possible roles of fructose 2,6-bisphosphate, fructose 6-phosphate, and phosphate in the control of PFP are discussed.     

Substrate specificity of pyrophosphate:fructose 6-phosphate 1-phosphotransferase from potato tuber

The aim of this work was to establish the precise ionic form of the reactants used by pyrophosphate:fructose-6-phosphate phosphotransferase. The enzyme was purified to near-homogeneity from potato (Solanum tuberosum L.) tubers. Changes in enzyme activity when the pH of the assay and the concentration of fructose 6-phosphate, pyrophosphate, and magnesium are varied independently indicate that fructose 6-phosphate²⁻ and MgP₂O₇²⁻ are the reacting species in the glycolytic direction. Analogous experiments with fructose 1,6-bisphosphate, inorganic phosphate, and magnesium demonstrate that the enzyme uses fructose 1,6-bisphosphate⁴⁻, HPO₄²⁻, and Mg²⁺ in the gluconeogenic direction. The ionic species used in the glycolytic direction are comparable with those required by bacterial ATP-dependent phosphofructokinase. This is consistent with the proposal that the active site of pyrophosphate:fructose-6-phosphate phosphotransferase in plants is equivalent to that of the bacterial phosphofructokinase (SM Carlisle et al. [1990] J Biol Chem 265: 18366-18371).     

A kinetic study of pyrophosphate: fructose-6-phosphate phosphotransferase from potato tubers. Application to a microassay of fructose 2,6-bisphosphate

Pyrophosphate : fructose-6-phosphate phosphotransferase (PPi-PFK) has been purified 150-fold from potato tubers and the kinetic properties of the purified enzyme have been investigated both in the forward and the reverse direction. Saturation curves for fructose 6-phosphate and also for fructose 1,6-bisphosphate were sigmoidal whereas those for PPi and Pi were hyperbolic. In the presence of fructose 2,6-bisphosphate, the affinity for fructose 6-phosphate and for fructose 1,6-bisphosphate were greatly increased and the kinetics became Micha?lian. The effect of fructose 2,6-bisphosphate was increased by the presence of fructose 6-phosphate and decreased by the presence of Pi. Consequently, the Ka for fructose 2,6-bisphosphate was as low as 5 nM for the forward reaction and reached 150 nM for the reverse reaction. On the basis of these properties, a procedure allowing one to measure fructose 2,6-bisphosphate in amounts lower than a picomole, is described.     

Inorganic pyrophosphate: fructose-6-phosphate 1-phosphotransferase of the potato tuber is related to the major ATP-dependent phosphofructokinase of E. coli

A procedure was developed for the purification of inorganic pyrophosphate: fructose-6-phosphate 1-phospho-transferase (PPi-PFK) from potato tubers. The enzyme has the structure alpha 4 beta 4 with a subunit of 68 kDa and a beta subunit of 60 kDa. The structural relationship of this enzyme to other PFKs and to fructose bisphosphatase was examined by immunoprecipitation and immunoblotting. Antibodies to the plant enzyme did not react with E. coli PFK. No cross-reaction was seen among the following enzymes or their antibodies: yeast fructose bisphosphatase; rabbit PFKs A, B, or the enzyme from brain; and the two subunits of the potato PPi-PFK. On the other hand, antibody to E. coli PFK-1 strongly cross-reacts with the 60 kDa polypeptide but not 68 kDa peptide.     

Distribution of pyrophosphate:fructose 6-phosphate phosphotransferase in maize leaves

In leaves of maize (Zea mays) the activity of pyrophosphate:fructose 6-phosphate 1-phosphotransferase (PFP) is much less than that of ATP:fructose 6-phosphate 1-phosphotransferase. A sequential extraction technique was used to study the location of PFP in this tissue. When compared with enzymes known to be restricted to specific locations in maize, the distribution of PFP activity in the sequential extracts indicated that PFP is located predominantly, if not exclusively, in the mesophyll cytoplasm. Although confined to the same site as sucrose synthesis, the level of PFP activity is inadequate to contribute significantly to the gluconeogenic flux from fructose 1,6-bisphosphate to fructose 6-phosphate. The absence of PFP activity from the bundle-sheath demonstrates that this activity is not essential for glycolysis in higher plants.     

Transgenic potato plants with strongly decreased expression of pyrophosphate:fructose-6-phosphate phosphotransferase show no visible phenotype and only minor changes in metabolic fluxes in their tubers

Potato (Solanum tuberosum L.) plants were transformed with antisense constructs to the genes encoding the -and -subunits of pyrophosphate: fructose-6-phosphate phosphotransferase (PEP), their expression being driven by the constitutive CaMV 35S promotor. (i) In several independent transformant lines, PFP expression was decreased by 70–90% in growing tubers and by 88–99% in stored tubers. (ii) The plants did not show any visual phenotype, reduction of growth or decrease in total tuber yield. However, the tubers contained 20–40% less starch than the wild type. Sucrose levels were slightly increased in growing tubers, but not at other stages. The rates of accumulation of sucrose and free hexoses when tubers were stored at 4° C and the final amount accumulated were the same in antisense and wild-type tubers. (iii) Metabolites were investigated at four different stages in tuber life history; growing (sink) tubers, mature tubers, cold-sweetening tubers and sprouting (source) tubers. At all stages, compared to the wild type, antisense tubers contained slightly more hexose-phosphates, two- to threefold less glycerate-3-phosphate and phosphoenolpyruvate and up to four-to fivefold more fructose-2,6-bisphosphate. (iv) There was no accumulation or depletion of inorganic pyrophosphate (PPi), or of UDP-glucose relative to the hexose-phosphates. (v) The pyruvate content was unaltered or only marginally decreased, and the ATP/ADP ratio did not change. (vi) Labelling experiments on intact tubers did not reveal any significant decrease in the unidirectional rate of metabolism of [U-¹⁴C]sucrose to starch, organic acids or amino acids. Stored tubers with an extreme (90%) reduction of PFP showed a 25% decrease in the metabolism of [U¹⁴-C] sucrose. (vii) Metabolism (cycling) of [U-¹⁴C]glucose to surcrose increased 15-fold in discs from growing antisense tubers, compared with growing wild-type tubers. Resynthesis of sucrose was increased by 10–20% when discs from antisense and wild-type tubers stored at 4° C (cold sweetening) were compared. The conversion of [U-¹⁴C]glucose to starch was decreased by about 30% and 50%, respectively. (viii) The randomisation of [1-¹³C]glucose in the glucosyl and fructosyl moieties of sucrose was decreased from 13.8 and 15.7% in the wild type to 3.6 and 3.9% in an antisense transformant. Simultaneously, randomisation in glucosyl residues isolated from starch was reduced from 14.4 to 4.1%. (ix) These results provide evidence that PFP catalyses a readily reversible reaction in tubers, which is responsible for the recycling of label from triose-phosphates to hexose-phosphates, but with the net reaction in the glycolytic direction. The results do not support the notion that PFP is involved in regulating the cytosolic PPi concentration. They also demonstrate that PFP does not control the rate of glycolysis, and that tubers contain exessive capacity to phosphorylate fructose-6-phosphate. The decreased concentration of phosphoenolpyruvate and glycerate-3-phosphate compensates for the decrease of PFP protein by stimulating ATP-dependent phosphofructokinase, and by stimulating fructose-6-phosphate,2-kinase to increase the fructose-2,6-bisphosphate concentration and activate the residual PFP. The decreased starch accumulation is explained as an indirect effect, caused by the increased rate of resynthesis (cycling) of sucrose in the antisense tubers.Abbreviations Fru1,6bisP fructose-1,6-bisphosphate - Fru2,6bisP fructose-2,6-bisphosphate - Fru6P fructose-6-phosphate - Glc1P glucose-1-phosphate - Glc6P glucose-6-phosphate - NMR nuclear magnetic resonance - 3PGA glycerate-3-phosphate - PEP phosphoenolpyruvate - PEP pyrophosphate: fructose-6-phosphate phosphotransferase - PFK phosphofructokinase - UDPGlc UDP glucose - WT wild type This research was supported by the Bundesministerium for Forschung and Technology (M.S., U.S.), the Canadian Research Council (S.C., D.D.), the Agricultural and Food Research Council (R.V.) and Sandoz Agro Ltd. (M.H., M.S.).     

A source of apparent pyrophosphate:fructose 6-phosphate phosphotransferase activity in rabbit muscle phosphofructokinase

In the presence of UDPglucose, rabbit muscle phosphofructokinase appeared to use PPi as a phosphoryl donor, as reported previously (Biochem. Biophys. Res. Commun. 121, 842-847). This apparent activity was due to conversion of UDPglucose and PPi to glucose 1-phosphate and UTP, the latter being metabolized by phosphofructokinase. Auxiliary enzymes used in the assays were contaminated by UDPglucose pyrophosphorylase. This contamination was sufficient to account for, and had similar properties to, the apparent PPi-dependent activity. Without auxiliary enzymes phosphofructokinase could not use PPi. These findings indicate that the apparent interconversion of phosphofructokinase and PPi:fructose 6-phosphate phosphotransferase must be re-assessed.     

Purification and properties of two fructose-6-phosphate phosphoketolases in Bifidobacterium

Fructose-6-phosphate phosphoketolase was purified from type strains of two species of the genus Bifidobacterium: B. globosum and B. dentium. The first species has a preferred animal habitat, like feces of animals and rumen of cattle; the latter is harboured in human habitats, like feces and dental caries of man. Two electrophoretic types of phosphoketolase (F6PPK) were previously distinguished and called animal and human type according to the habitat of the bifid organism. The purified preparations of these two phosphoketolases displayed very different optimum pH range, metal activator and molecular weight; outstanding difference was found in the substrate specificity: the enzyme from B. globosum was able to split xylulose-5-P as well as fructose-6-P, whereas the phosphoketolase from B. dentium appeared to be specific for fructose-6-P.     

Binding of fructose-6-phosphate to phosphofructokinase from yeast.

Yeast phosphofructokinase binds one molecule of fructose-6-phosphate per subunit. The binding curve exhibits sigmoidality and yields a good fit to an equation derived from the kinetic model as developed previously for this enzyme. The results show that the allosteric kinetic response of the enzyme to fructose-6-phosphate is due to cooperativity of the binding process.     

Glucose 6-phosphate dehydrogenase from sweet potato

Glucose 6-phosphate dehydrogenase was purified about 290-foldfrom sweet potato root tissue. The molecular weight was estimatedto be 110,000 by Bio-Gel 300 column chromatography. A LINEWEAVER-BURKplot of the reciprocal rate against reciprocal glucose 6-phosphateconcentration was concave downwards. A HILL coefficient lessthan 1 was obtained at lower concentrations of glucose 6-phosphate(below 0.5 mM). These results suggest that binding of glucose6-phosphate to the enzyme occurs with negative cooperativity. (Received April 30, 1970; )     

Studies of the structure of fructose-6-phosphate 2-kinase:fructose-2,6-bisphosphatase

Some physicochemical properties of a homogeneous preparation of a bifunctional enzyme, fructose-6-phosphate 2-kinase:fructose-2,6-bisphosphatase, were determined. The molecular weight of the enzyme is 101 000 as determined by high-speed sedimentation equilibrium. The molecular weight of dissociated enzyme is 55 000 in 6 M guanidinium chloride by sedimentation equilibrium and in sodium dodecyl sulfate by polyacrylamide gel electrophoresis. A value of 4.7 was observed for the isoelectric point. Tryptic peptide maps and high-performance liquid chromatography of the trypsin-digested enzyme revealed approximately 60 peptides. Amino acid analysis of the enzyme shows that it contains 27 lysine and 36 arginine residues per 55 000 daltons. No free N-terminal amino acid residue was detectable, suggesting that it is blocked. Hydrolysis of the enzyme by carboxypeptidases A and B releases tyrosine followed by histidine and arginine, indicating that the amino acid sequence at the carboxyl terminus is probably -Arg-His-Tyr. Tryptic digestion of [32P]phosphofructose-6-phosphate 2-kinase:fructose-2,6-bisphosphatase yields a 32P-labeled peptide detected by tryptic peptide mapping and high-performance liquid chromatography. Thermolysin digestion of CNBr-cleaved 32P-enzyme also yields a single 32P-peptide. These results indicate that fructose-6-phosphate 2-kinase:fructose-2,6-bisphosphatase is a dimer of 55 000 daltons and the subunits are very similar, if not identical.     

Isomerase activity of the C-terminal fructose-6-phosphate binding domain of glucosamine-6-phosphate synthase from Escherichia coli

The isomerase activity of the C-terminal fructose-6P binding domain (residues 241-608) of glucosamine-6-phosphate synthase from Escherichia coli has been studied. The equilibrium constant of the C-terminal domain k(eq) ([glucose-6P]/[fructose-6-P]) = 5.0. A non-competitive product inhibition of the isomerase activity by the reaction product glucose-6-P has been detected. The existence of more than one binding and reaction sites for the substrate fructose-6P on the molecule of glucosamine-6-phosphate synthase can be expected. The fructose-6P binding domain possibly includes a regulatory site, different from the catalytic center of the enzyme.     

Molecular characterization of four forms of phosphofructokinase purified from potato tuber

Four forms of phosphofructokinase (PFK) have been purified to apparent homogeneity from tubers of potato (Solanum tuberosum cv. Record). Each had a final specific activity of about 200 mumol.min-1.mg-1 protein. Similar forms of PFK were found in partially purified extracts from tubers and leaves of other potato cultivars and related wild species. The relative molecular masses of three forms of PFK were about 200,000 whereas that of the fourth PFK was greater than 800,000. The four forms of PFK contained different proportions of four polypeptides which had apparent relative molecular masses of 46,300, 49,500, 50,000, and 53,000. These polypeptides gave different patterns of peptide fragments after chemical and proteolytic cleavage. Western blots and immunoprecipitation studies using antibodies raised against the individual polypeptides showed that all four are associated with PFK. Thus, potato tubers contain four distinct forms of PFK that differ in their subunit composition.     

The murine glutamine:fructose-6-phosphate amidotransferase-encoding cDNA sequence

Glutamine:fructose-6-phosphate amidotransferase (GFAT) is the rate-limiting enzyme in hexosamine synthesis and has been implicated in the control of growth factor gene expression. We cloned a mouse cDNA which is 91% homologous to the human sequence. The deduced amino-acid sequence shows 98.6% identity to human GFAT. The cDNA is derived from a 7-kb mRNA in the mouse, while there are multiple-sized human mRNAs.