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.     

Molecular properties of pyrophosphate:fructose-6-phosphate phosphotransferase from potato tuber

Pyrophosphate:fructose-6-phosphate phosphotransferase (PFP) from potato tubers has been purified to homogeneity. The enzyme contains two polypeptides with apparent relative molecular mass (Mr) values of 65,000 and 60,000. These polypeptides give different peptide fragments after limited proteolytic digestion. Antibodies raised against each polypeptide separately are specific for that polypeptide, but both antisera are capable of immunoprecipitating native PFP activity. These antibodies also recognize similar pairs of polypeptides in a range of other plant tissues that contain PFP activity. Based on gel filtration, the Mr value of potato tuber PFP is 265,000. This suggests that the enzyme is a heterotetramer composed of two polypeptides with Mr values of 65,000 and 60,000. In the presence of pyrophosphate, potato PFP dissociates into a 130,000 dimer.     

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.     

Redox active sulfhydryls are required for fructose 2,6-bisphosphate activation of plant pyrophosphate fructose-6-phosphate 1-phosphotransferase.

The classical, alpha/beta-subunit form (Q2) of green tomato pyrophosphate fructose-6-phosphate 1-phosphotransferase (PFP, EC 2.7.1.90), a cytosolic enzyme functional in carbohydrate metabolism, was rapidly inactivated on incubation with the oxidant 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Analysis of the DTNB-treated sample by a fluorescence procedure revealed that inactivation was accompanied by oxidation of sulfhydryl groups, primarily on the alpha-subunit. Phosphate metabolites--fructose 2,6-bisphosphate, fructose 1,6-bisphosphate, Pi, and PPi--protected against DTNB inactivation to varying degrees. The Km values for fructose 6-phosphate and PPi were not changed by DTNB treatment, but the capability for activation by fructose 2,6-bisphosphate was severely diminished. The oxidative inactivation of PFP was reversed by dithiothreitol, but not by monothiols (reduced glutathione or beta-mercaptoethanol). Reactivation was accompanied by restoration of the ability to undergo activation by fructose 2,6-bisphosphate. The findings suggest that sulfhydryl groups are essential for the activation of PFP by fructose 2,6-bisphosphate and raise the possibility that a reversible change in their redox status can take place under certain conditions. Evidence that this is the case was obtained with a preparation from wheat flour which, in the absence of an added oxidant, required reduction by a dithiol for activation by fructose 2,6-bisphosphate (dithiothreitol and reduced thioredoxin h).     

Fructose 2,6-bisphosphate as a contaminant of commercially obtained fructose 6-phosphate: Effect on PPi: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.     

Multiple forms of pyrophosphate:D-fructose-6-phosphate 1-phosphotransferase from wheat seedlings. Regulation by fructose 2,6-bisphosphate

Two forms of pyrophosphate:D-fructose-6-phosphate 1-phosphotransferase have been isolated from wheat seedlings. One of these enzymes, termed PFP-1, has been purified to homogeneity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the enzyme is composed of two different polypeptide chains of Mr = 67,000 (alpha) and 60,000 (beta). PFP-1 has been assigned a molecular structure consisting of alpha 2 beta 2 based on an estimated Mr of 234,000 for the native enzyme. PFP-2, the other form of phosphotransferase, has also been purified extensively. Preliminary data suggest that the active form of PFP-2 is probably a dimer of a polypeptide chain of Mr = 60,000. Immunological studies indicate that the two enzyme preparations share common antigenic determinants. The two forms of enzyme have very similar kinetic properties. The phosphotransferases are activated by fructose 2,6-bisphosphate (Fru-2,6-P2) which lowers the Km of the enzymes for fructose 6-phosphate but not that for PPi. Interestingly, PFP-1 is significantly more active than PFP-2 in the absence of Fru-2,6-P2. Also, PFP-1 exhibits a greater affinity (Ka = 7 nM) than PFP-2 (Ka = 26 nM) for the activator. Based on kinetic, immunological, and physicochemical parameters, it is suggested that the two enzymic forms are related in that they share the same catalytic moiety, i.e. the 60,000-dalton or beta subunit. The beta subunit when in complex formation with the alpha subunit, as in PFP-1, becomes more active in the absence of Fru-2,6-P2 as well as exhibits a greater sensitivity toward the effector.     

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.     

Physiological relevance of fructose 2,6-bisphosphate in the regulation of spinach leaf pyrophosphate:fructose 6-phosphate 1-phosphotransferase

A major problem in defining the physiological role of pyrophosphate:fructose 6-phosphate 1-phosphotransferase (PFP, EC 2.7.1.90) is the 1,000-fold discrepancy between the apparent affinity of PFP for its activator, fructose 2,6-bisphosphate (Fru-2,6-P₂), determined under optimum conditions in vitro and the estimated concentration of this signal metabolite in vivo. The aim of this study was to investigate the combined influence of metabolic intermediates and inorganic phosphate (Pi) on the activation of PFP by Fru-2,6-P₂. The enzyme was purified to near-homogeneity from leaves of spinach (Spinacia oleracea L.). Under optimal in vitro assay conditions, the activation constant (K _a) of spinach leaf PFP for Fru-2,6-P₂ in the glycolytic direction was 15.8 nM. However, in the presence of physiological concentrations of fructose 6-phosphate, inorganic pyrophosphate (PPi), 3-phosphoglycerate (3PGA), phosphoenolpyruvate (PEP), ATP and Pi the K _a of spinach leaf PFP for Fru-2,6-P₂ was up to 2000-fold greater than that measured in the optimised assay and V _max decreased by up to 62%. Similar effects were observed with PFP purified from potato (Solanum tuberosum L.) tubers. Cytosolic metabolites and Pi also influenced the response of PFP to activation by its substrate fructose 1,6-bisphosphate (Fru-1,6-P₂). When assayed under optimum conditions in the gluconeogenic direction, the K _a of spinach leaf PFP for Fru-1,6-P₂ was approximately 50 μM. Physiological concentrations of PPi, 3PGA, PEP, ATP and Pi increased K _a up to 25-fold, and decreased V _max by over 65%. From these results it was concluded that physiological concentrations of metabolites and Pi increase the K _a of PFP for Fru-2,6-P₂ to values approaching the concentration of the activator in vivo. Hence, measured changes in cytosolic Fru-2,6-P₂ levels could appreciably alter the activation state of PFP in vivo. Moreover, the same levels of metabolites increase the K _a of PFP for Fru-1,6-P₂ to an extent that activation of PFP by this compound is unlikely to be physiologically relevant. Received: 21 July 2000 / Accepted: 15 September 2000     

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.     

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.     

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).     

Oscillation in fructose 2,6-bisphosphate levels and in the phosphorylation states of fructose 6-phosphate,2-kinase:fructose-2,6-bisphosphatase in ischemic rat liver.

In order to determine the role of fructose (Fru) 2,6-P2 in stimulation of phosphofructokinase in ischemic liver, tissue contents of Fru-2,6-P2, hexose-Ps, adenine nucleotides, and Fru-6-P,2-kinase:Fru-2,6-bisphosphatase were investigated during the first few minutes of ischemia. The Fru-2,6-P2 concentration in the liver changed in an oscillatory manner. Within 7 s after the initiation of ischemia, Fru-2,6-P2 increased from 6 to 21 nmol/g liver and decreased to 5 nmol/g liver within 30 s. Subsequently, it reached the maximum value at 50, 80, and 100 s and decreased to the basal concentration at 60, 90, and 120 s. Oscillatory patterns were also observed with Glc-6-P and Fru-6-P, but the ATP/ADP ratio decreased monotonically. Determination of Fru-6-P,2-kinase activity and the phosphorylation states of Fru-6-P,2-kinase:Fru-2,6-bisphosphatase demonstrated that at 7 and 50 s, where Fru-2,6-P2 was the highest, the enzyme was activated and mostly in a dephosphorylated form. On the other hand, at 0, 30, and 300 s, the enzyme was predominantly in the phosphorylated form. The concentration of cAMP in the liver also changed in an oscillatory manner between 0.5 to 1.3 nmol/g with varying frequency of 10 to 40 s. These results indicated that: (a) Fru-2,6-P2 was important in rapid activation of phosphofructokinase in the first few seconds and up to 2-3 min, and (b) the oscillation of Fru-2,6-P2 concentration was the result of activation and inhibition of Fru-6-P,2-kinase:Fru-2,6-bisphosphatase, which was caused by changes in the phosphorylation state of the enzyme.     

Phosphofructokinase 2: the enzyme that forms fructose 2,6-bisphosphate from fructose 6-phosphate and ATP

The polypeptide composition of plasmalemma, mitochondria and endoplasmic reticulum, isolated from maize coleoptile, was determined using polyacrylamide gel electrophoresis in the presence of lithium dodecyl sulfate. A polypeptide with an apparent molecular weight of 90,000 was found to be the major polypeptide associated with plasmalemma. This polypeptide is firmly attached to the membrane since it was not extracted by KCl. However, after a treatment with Triton X100, the polypeptide was detached from the membrane. It was also found that a heat treatment of the membrane partly destroyed this polypeptide. This effect of heat treatment was also observed after isolation of the polypeptide by electrophoresis.     

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.     

Fructose 2,6-bisphosphate activates pyrophosphate: fructose-6-phosphate 1-phosphotransferase and increases triose phosphate to hexose phosphate cycling in heterotrophic cells

The aim of this work was to establish the influence of fructose 2,6-bisphosphate (Fru-2,6-P₂) on non-photosynthetic carbohydrate metabolism in plants. Heterotrophic callus lines exhibiting elevated levels of Fru-2,6-P₂ were generated from transgenic tobacco (Nicotiana tabacum L.) plants expressing a modified rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Lines containing increased amounts of Fru-2,6-P₂ had lower levels of hexose phosphates and higher levels of 3-phosphoglycerate than the untransformed control cultures. There was also a greater redistribution of label into the C6 position of sucrose and fructose, following incubation with [1-¹³C]glucose, in the lines possessing the highest amounts of Fru-2,6-P₂, indicating a greater re-synthesis of hexose phosphates from triose phosphates in these lines. Despite these changes, there were no marked differences between lines in the metabolism of ¹⁴C-substrates, the rate of oxygen uptake, carbohydrate accumulation or nucleotide pool sizes. These data provide direct evidence that physiologically relevant changes in the level of Fru-2,6-P₂ can affect pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP) activity in vivo, and are consistent with PFP operating in a net glycolytic direction in the heterotrophic culture. However, the results also show that activating PFP has little direct effect on heterotrophic carbohydrate metabolism beyond increasing the rate of cycling between hexose phosphates and triose phosphates. Received: 29 March 2000 / Accepted: 13 June 2000     

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.).     

Regulation of the fructose 6-phosphate/fructose 2,6-bisphosphate cycle by enzyme phosphorylation and sn-glycerol 3-phosphate

The regulation of the Fru-6-P/Fru-2,6-P2 cycle by the cooperation of allosteric and covalent mechanisms was investigated in a reconstituted enzyme system under in vitro conditions. Phosphorylation of the bifunctional enzyme exerts a much stronger effect than sn-glycerol 3-phosphate in lowering the quasi-stationary concentration of fructose 2,6-bisphosphate and in increasing the critical concentration of the fructose phosphates, respectively. However, sn-glycerol 3-phosphate is able to strongly amplify the decrease of the quasi-stationary concentration of fructose 2,6-bisphosphate due to phosphorylation. The experiments can be described by a mathematical model involving rate equations for the dephosphorylated and the phosphorylated PFD-2 and FBPase-2. The results are compared with data from the literature obtained under in vivo conditions.     

Fluorescence study of ligand binding to potato tuber pyrophosphate-dependent phosphofructokinase: evidence for competitive binding between fructose-1,6-bisphosphate and fructose-2,6-bisphosphate

The intrinsic fluorescence of potato tuber pyrophosphate:fructose-6-phosphate 1-phosphotransferase (PFP) was used as an indicator of conformational changes due to ligand binding. Binding of the substrates and the allosteric activator fructose-2,6-bisphosphate was quantitatively compared to their respective kinetic effects on enzymatic activity. PFP exhibited a relatively high affinity for its isolated substrates, relative to the enzyme's respective K(m) (substrate) values. There are two distinct types of fructose-1,6-bisphosphate interaction with PFP, corresponding to catalytic and activatory binding. Activatory fructose-1,6-bisphosphate binding shares several characteristics with fructose-2,6-bisphosphate binding, indicating that both ligands compete for the same allosteric activator site. Activation by fructose-1,6-bisphosphate or fructose-2,6-bisphosphate was exerted primarily on the forward (glycolytic) reaction by greatly increasing the enzyme's affinity for fructose-6-phosphate. Binding of substrates and effectors to PFP and PFP kinetic properties were markedly influenced by assay pH. Results indicate an increased glycolytic role for PFP during cytosolic acidification that accompanies anoxia stress.     

Catalytic site of rat liver and bovine heart fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase. Identification of fructose 6-phosphate binding site

Fructose-6-P binding sites of rat liver and bovine heart Fru-6-P,2-kinase:Fru-2,6-bisphosphatase were investigated with an affinity labeling reagent, N-bromoacetylethanolamine phosphate. The rat liver enzyme was inactivated 97% by the reagent in 60 min, and the rate of inactivation followed pseudo-first order kinetics. The bovine heart enzyme was inactivated 90% within 60 min, but the inactivation rate followed pseudo-first order up to 80% inactivation and then became nonlinear. The presence of fructose-6-P retarded the extent of the inactivation to approximately 40% in 60 min. In order to determine the amino acid sequence of the fructose-6-P binding site, both enzymes were reacted with N-bromo[14C]acetylethanolamine-P and digested with trypsin; radiolabeled tryptic peptides were isolated and sequenced. A single 14C-labeled peptide was isolated from the rat liver enzyme, and the amino acid sequence of the peptide was determined as Lys-Gln-Cys-Ala-Leu-Ala-Leu-Lys. A major and two minor peptides were isolated from bovine heart enzyme whose amino acid sequences were Lys-Gln-Cys-Ala-Leu-Val-Ala-Leu-Lys, Arg-Ile-Glu-Cys-Tyr-Lys, and Ile-Glu-Cys-Tyr-Lys, respectively. In all cases, N-bromoacetylethanolamine-P had alkylated the cysteine residues. The amount of bromo[14C]acetylethanolamine-P incorporated into rat liver and beef heart was 1.3 mol/mol of subunit and 2.1 mol/mol of subunit, respectively, and the incorporations in the presence of Fru-6-P were reduced to 0.34 mol/mol of subunit and 0.9 mol/mol of subunit, respectively. Thus, the main fructose-6-P binding site of rat liver and bovine heart enzymes was identical except for a single amino acid substitution of valine for alanine in the latter enzyme. This peptide corresponded to residues 105 to 113 from the N terminus of the known amino acid sequence of rat liver enzyme, but since the complete sequence of bovine heart enzyme is not known, the location of the same peptide in the heart enzyme cannot be assigned.     

Activation of pyrophosphate:fructose-6-phosphate 1-phosphotransferase by fructose 2,6-bisphosphate stimulates conversion of hexose phosphates to triose phosphates but does not influence accumulation of carbohydrates in phosphate-deficient tobacco cells

The aim of this work was to investigate the contribution of fructose 2,6-bisphosphate to the regulation of carbohydrate metabolism under phosphate stress. The study exploited heterotrophic tobacco callus lines expressing a modified mammalian 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase that increased the fructose 2,6-bisphosphate content of the tissue. The phosphate status of two transgenic and one untransformed cell line was perturbed by incubation with 2-deoxyglucose, a phosphate-sequestering agent, and by growth of callus on phosphate-depleted media. 31P-NMR spectroscopy confirmed that both treatments decreased cellular levels of inorganic phosphate and phosphorylated metabolites. Despite large decreases in the amounts of phosphate esters, UDPglucose and adenylates in response to phosphate deficiency, the fructose 2,6-bisphosphate content of each line was unaffected by 2-deoxyglucose and increased during growth on phosphate-limited media. Short-term treatment of callus with 2-deoxyglucose had only minor effects on the carbohydrate status of each line, whereas long-term phosphate deficiency caused an increase in starch and a decrease in soluble sugar content in both transgenic and control lines. There were no consistent differences between the three callus lines in metabolism of [U-14C]glucose in response to incubation with 2-deoxyglucose. In contrast, there was a decrease in partitioning of label into glycolytic products (particularly organic acids) in untransformed callus during growth on phosphate-depleted medium. This decrease was greatly attenuated in the transgenic lines with increased fructose 2,6-bisphosphate content. This suggests that the conversion of hexose phosphates to triose phosphates is constrained under phosphate-deficient conditions, and that this restriction can be relieved by activation of pyrophosphate:fructose-6-phosphate 1-phosphotransferase. However, since the transgenic and control lines did not differ in the extent to which the carbohydrate content changed in response to growth on phosphate-depleted media, it is concluded that an increase in flux through pyrophosphate:fructose-6-phosphate 1-phosphotransferase is not a major component of the metabolic response of heterotrophic tobacco cells to phosphate deficiency.