Fructose 2,6-bisphosphate. A new activator of phosphofructokinase

A new activator of rat liver phosphofructokinase was partially purified from rat hepatocyte extracts by DEAE-Sephadex chromatography. The activator, which eluted in the sugar diphosphate region, was sensitive to acid treatment but resistant to heating in alkali. Mild acid hydrolysis resulted in the appearance of a sugar monophosphate which was identified as fructose 6-phosphate by gas chromatography/mass spectroscopy. These observations suggest that the activator is fructose 2,6-bisphosphate. This compound was synthesized by first reacting fructose 1,6-bisphosphate with dicyclohexylcarbodiimide and then treating the cyclic intermediate with alkali. The structure of the synthetic compound was definitively identified as fructose 2,6-bisphosphate by 13C NMR spectroscopy. Fructose 2,6-bisphosphate had properties identical with those of the activator purified from hepatocyte extracts. It activated both the rat liver and rabbit skeletal muscle enzyme in the 0.1 microM range and was several orders of magnitude more effective than fructose 1,6-bisphosphate. Fructose 2,6-bisphosphate was not a substrate for aldolase or fructose 1,6-bisphosphatase. It is likely that this new activator is an important physiologic factor of phosphofructokinase in vivo.     

The activities of fructose diphosphatase in flight muscles from the bumble-bee and role of this enzyme in heat generation

1. The maximum catalytic activities of fructose diphosphatase from flight muscles of bumble-bees (Bombus spp.) are at least 30-fold those reported for the enzyme from other tissues. The maximum activity of fructose diphosphatase in the flight muscle of any particular bee is similar to that of phosphofructokinase in the same muscle, and the activity of hexokinase is similar to or greater than the activity of phosphofructokinase. There is no detectable activity of glucose 6-phosphatase and only a very low activity of glucose 6-phosphate dehydrogenase in these muscles. The activities of both fructose diphosphatase and phosphofructokinase vary inversely with the body weight of the bee, whereas that of hexokinase is relatively constant. 2. There is no significant hydrolysis of fructose 1-phosphate, fructose 6-phosphate, glucose 1,6-diphosphate and glycerol 3-phosphate by extracts of bumble-bee flight muscle. 3. Fructose 1,6-diphosphatase from bumble-bee flight muscle and from other muscles is inhibited by Mn(2+) and univalent cations; the potency of inhibition by the latter varies in the order Li(+)>Na(+)>K(+). However, the fructose diphosphatase from bumble-bee flight muscle is different from the enzyme from other tissues in that it is not inhibited by AMP. 4. The contents of ATP, hexose monophosphates, fructose diphosphate and triose phosphates in bumble-bee flight muscle showed no significant changes between rest and flight. 5. It is proposed that both fructose diphosphatase and phosphofructokinase are simultaneously active and catalyse a cycle between fructose 6-phosphate and fructose diphosphate in resting bumble-bee flight muscle. Such a cycle would produce continuous hydrolysis of ATP, with the release of energy as heat, which would help to maintain the thoracic temperature during rest periods at a level adequate for flight.     

Fructose syrups and ethanol production by selective fermentation of inulin

Jerusalem artichoke is a favorable substrate for inulin or fructose syrup production. The sugar content and the fructose ratio of inulin depend on various factors, particularly on the date of harvest. Incomplete fermentation of extracts by selected yeasts allows the production of inulin with increased fructose content. The yeast strains (Saccharomyces cerevisiae, S. diastaticus...) are chosen for their ability to ferment sucrose and inulin small polymers, but not easily inulin large polymers. A good increase in the fructose ratio and a good yield in residual sugars can be obtained with the better strains. After fermentation and acid or enzymatic hydrolysis, extracts from early and late harvested tubers lead to syrups of good quality containing up to 95% and 90% of fructose respectively. This fermentative enrichment process is competitive with others (for example, chromatographic enrichment), is appropriate to raw extracts, simplifies the purification steps, and also permits the simultaneous benefit of production of by-products in the form of ethanol and yeast (in addition to the pulps). Unhydrolyzed inulin polymers with high fructose content can be recovered by this selective fermentation.     

Fractionation and characterization of the phosphoenolpyruvate: fructose 1-phosphotransferase system from Pseudomonas aeruginosa

The initial reactions involved in the catabolism of fructose in Pseudomonas aeruginosa include the participation of a phosphoenolpyruvate:fructose 1-phosphotransferase system (F-PTS). Fractionation of crude extracts of fructose-grown cells revealed that both membrane-associated and soluble components were essential for F-PTS activity. Further resolution of the soluble fraction by both size exclusion and ion-exchange chromatography revealed the presence of only one component, functionally analogous to enzyme I. Enzyme I exhibited a relative molecular weight of 72,000, catalyzed the pyruvate-stimulated hydrolysis of phosphoenolpyruvate to pyruvate, and mediated the phosphorylation of fructose when combined with a source of enzyme II (washed membranes). No evidence for the requirement of a phosphate carrier protein, such as HPr, could be demonstrated. Thus, the F-PTS requires a minimum of two components, a soluble enzyme I and a membrane-associated enzyme II complex, and both were shown to be inducible. Reconstituted F-PTS activity was specific for phosphoenolpyruvate as a phosphate donor (Km, approximately -0.6 mM) and fructose as the sugar substrate (Km, approximately 18 microM). Components of the Pseudomonas F-PTS did not restore activity to extracts of deletion mutants of Salmonella typhimurium deficient in individual proteins of the PTS or to fractionated membrane and soluble components of the F-PTS of Escherichia coli. Similarly, membrane and soluble components of E. coli and S. typhimurium would not cross-complement the F-PTS components from P. aeruginosa.     

Regulation of rat liver fructose 2,6-bisphosphatase

An enzyme activity that catalyzes the hydrolysis of phosphate from the C-2 position of fructose 2,6-bisphosphate has been detected in rat liver cytoplasm. The S0.5 for fructose 2,6-bisphosphate was about 15 microM and the enzyme was inhibited by fructose 6-phosphate (Ki 40 microM) and activated by Pi (KA 1 mM). Fructose 2,6-bisphosphatase activity was purified to homogeneity by specific elution from phosphocellulose with fructose by specific elution from phosphocellulose with fructose 6-phosphate and had an apparent molecular weight of about 100,000, 6-phosphofructo 2-kinase activity copurified with fructose 2,6-bisphosphatase activity at each step of the purification scheme. Incubation of the purified protein with [gamma-32P]ATP and the catalytic subunit of the cAMP-dependent protein kinase resulted in the incorporation of 1 mol of 32P/mol of enzyme subunit (Mr = 50,000). Concomitant with this phosphorylation was an activation of the fructose 2,6-bisphosphatase and an inhibition of the 6-phosphofructo 2-kinase activity. Glucagon addition to isolated hepatocytes also resulted in an inhibition of 6-phosphofructo 2-kinase and activation of fructose 2,6-bisphosphatase measured in cell extracts, suggesting that the hormone regulates the level of fructose 2,6-bisphosphate by affecting both synthesis and degradation of the compound. These findings suggest that this enzyme has both phosphohydrolase and phosphotransferase activities i.e. that it is bifunctional, and that both activities can be regulated by cAMP-dependent phosphorylation.     

Initial characterization of sucrose-6-phosphate hydrolase from Streptococcus mutans and its apparent identity with intracellular invertase.

An intracellular enzyme catalyzing the hydrolysis of sucrose-6-phosphate to glucose-6-phosphate and fructose has been identified in extracts of $\text{Streptococcus}\text{mutans}$ 6715-10. The preparation was purified chromatographically and found to have an apparent molecular weight of 42,000. The enzyme has as a K_m for sucrose-6-phosphate of 0.21 mM, a pH optimum of 7.1, is quite stable and requires no added cofactors or metal ions. Sucrose is a competitive inhibitor of sucrose-6-phosphate hydrolysis (K_i = 8. 12 mM). A previously described intracellular invertase copurifies with the enzyme and could not be separated from it by disc gel electrophoresis. It is concluded that intracellular invertase is a sucrose-6-phosphate hydrolase with a low catalytic activity for hydrolysis of sucrose.     

Influence of Yeast Flocculation on the Rate of Jerusalem Artichoke Extract Fermentation

Variations in residual sugar composition have been observed during Jerusalem artichoke extract fermentations by using Saccharomyces diastaticus NCYC 625, a flocculating yeast strain. In batch cultures, these differences were due to the inulin polymer size distribution of the extracts: measurements of enzymatic activities on different polymerized substrates have shown that the hydrolysis and fermentation yield decreased when the fructose/glucose ratio of the extract increased. Inulin hydrolysis appeared to be the limiting factor of the fermentation rate. A comparison of continuous and batch cultures with the same extract showed that fermentability differences were related to the structure and size of the yeast flocs. This led to an hydrolysis selectivity of the inulin polymers according to their size: the chemostat culture in which the floc average size was larger gave longer chained residual sugars. Received: 8 November 1999 / Accepted: 24 February 2000     

Kinetic studies on dextransucrase from the cariogenic oral bacterium Streptococcus mutans

The kinetic mechanism of dextransucrase was studied using the Streptococcus mutans enzyme purified by affinity chromatography to a specific activity of 36.9 mumol/min/mg of enzyme. In addition to dextran synthesis, the enzyme catalyzed sucrose hydrolysis and isotope exchange between fructose and sucrose. The rates of sucrose hydrolysis and dextran synthesis were partitioned as a function of dextran concentration such that exclusive sucrose hydrolysis was observed in the absence of dextran and exclusive dextran synthesis at high dextran concentrations. An analogous situation was observed with fructose-dependent partitioning of sucrose hydrolysis and fructose exchange. Steady state dextran synthesis and fructose isotope exchange kinetics were simplified by assay at dextran or fructose concentrations high enough to eliminate significant contributions from sucrose hydrolysis. This limited dextran synthesis assays to dextran concentrations above apparent saturation. The limitation was diminished by establishing conditions in which the enzyme does not distinguish between dextran as a substrate and product which allowed initial discrimination among mechanisms on the basis of the presence or absence of dextran substrate inhibition. No inhibition was observed, which excluded ping-pong and all but three common sequential mechanisms. Patterns of initial velocity fructose production inhibition and fructose isotope exchange at equilibrium were consistent with dextran synthesis proceeding by a rapid equilibrium random mechanism. A nonsequential segment was apparent in the exchange reaction between fructose and sucrose assayed in the absence of dextran. However, the absence of detectable glucosyl exchange between dextrans and the lack of steady state dextran substrate inhibition indicate that glucosyl transfer to dextran must occur almost exclusively through the sequential route. A review of the kinetic constants from steady state dextran synthesis, fructose product inhibition, and fructose isotope exchange showed a consistency in constants derived from each reaction and revealed that dextran binding increases the affinity of sucrose and fructose for dextransucrase.     

Fructose metabolism in four Pseudomonas species

1. ATP-Dependent phosphorylation of fructose could not be detected in extracts of fructose-grown cells of Pseudomonas extorquens strain 16, Pseudomonas 3A2, Pseudomonas acidovorans and Pseudomonas fluorescens. Instead, phosphorylation of fructose to fructose-1-phosphate was found to occur when cell-free extracts were incubated with fructose and phosphoenolpyruvate. Such an activity could not be detected in cell-free extracts of succinate-grown cells. 2. High levels of 1-phosphofructokinase were found in extracts of the above organisms when growth on fructose. 3. Mutants of Pseudomonas extorquens strain 16 lacking 1-phosphofructokinase were unable to grow on fructose. Revertants to growth on fructose had regained the capacity to synthesize this enzyme, indicating its necessary involvement in fructose metabolism. 4. A survey has been carried out of enzymes involved in carbohydrate metabolism in the species listed above.     

Alcohol fermentation of sweet potato. I. Acid hydrolysis and factors involved

Factors affecting acid hydrolysis of sweet potato powder (SSP) to fermentable sugars were examined. These include HCl concentration, temperature, time, and levels of SPP. Maximum reducing sugar, reported as dextrose equivalent (DE), was detected after 24 min hydrolysis (1% SPP) in 0.034N HCl heated at 154°C. These samples also had 3.43% droxymethylfurfural (HMF) based on dry weight. A high level of HMF (9.2%) was detected in 1% SPP heated at 154° in 0.10N HCl for 18 min. The lowest concentration of HMF formed (1.8%), at maximal DE of 61%, was established in samples containing 5% SPP and heated at 154° in 0.034N HCl for 48 min. Aqueous extracts of uncured SPP, examined by HPLC, contained glucose, fructose and sucrose, but degraded SPP had only glucose and fructose. Products of degraded SPP, under appropriate conditions, could be used for alcohol fermentation.     

Pyrophosphate-dependent sucrose metabolism and its activation by fructose 2,6-bisphosphate in sucrose importing plant tissues

In the presence of pyrophosphate and uridine diphosphate, sucrose was cleaved to form glucose 1-phosphate and fructose with soluble extracts from sucrose importing plant tissues. The glucose 1-phosphate then was converted through glycolysis to triose phosphates in a pyrophosphate-dependent pathway which was activated by fructose 2,6-bisphosphate. Much less activity, less than 5%, was found in sucrose exporting tissue extracts from the same plants. These findings suggest that imported sucrose is metabolized in the cytoplasm of plant tissues by utilizing pyrophosphate and that sucrose metabolism is partially regulated by fructose 2,6-bisphosphate.     

Unambiguous stereochemical course of rabbit liver fructose bisphosphatase hydrolysis

The stereochemical course of rabbit liver fructose bisphosphatase (EC 3.1.3.11) was determined by hydrolyzing the substrate analogue (Sp)-[1-18O]fructose 1-phosphorothioate 6-phosphate in H(2)17O, incorporating the chiral, inorganic phosphorothioate product into adenosine 5'-O-(2-thiotriphosphate) (ATP beta S), and analyzing the isotopic distribution of 18O in ATP beta S by 31P NMR. The result indicates that the 1-phosphoryl group is transferred with inversion of configuration. A series of single-turnover experiments ruled out an acyl phosphate intermediate in the hydrolysis. Consequently, fructose bisphosphatase catalyzes the hydrolysis of fructose 1,6-bisphosphate via a direct transfer of the phosphoryl moiety to water.     

The phosphoenolpyruvate-dependent fructose-specific phosphotransferase system in Rhodopseudomonas sphaeroides. Energetics of the phosphoryl group transfer from phosphoenolpyruvate to fructose

Energy coupling to fructose transport in Rhodopseudomonas sphaeroides is achieved by phosphorylation of the membrane-spanning fructose-specific carrier protein, EFruII. The phosphoryl group of phosphoenolpyruvate is transferred to EFruII via the cytoplasmic component SF (soluble factor). The standard free enthalpy of hydrolysis of the two phosphorylated proteins has been estimated from isotope exchange measurements in chemical equilibrium. The delta G degrees for SF-P is -60.5 kJ/mol. The standard free enthalpy for hydrolysis of EII-P is -37.9 kJ/mol, but -45.2 kJ/mol when SF is still complexed to it, as in the overall reaction. Therefore the standard free enthalpy of hydrolysis of SF X EII-P is 70% of the standard free enthalpy of hydrolysis of P-enolpyruvate. The measurements reveal two regulation sites in the system. First, the phosphorylation of SF is inhibited by pyruvate when the concentration ratio of pyruvate/P-enolpyruvate becomes too high. Second, a low concentration of internal fructose prevents the phosphorylation of the carrier by the internal fructose-1-P pool when the concentration of the latter becomes too high or the phosphorylation rate by P-enolpyruvate too slow. Furthermore comparison of the isotope exchange and the overall phosphotransferase reaction kinetics leads to the conclusion that binding of fructose to the carrier is a slow step relative to the phosphoryl group transfer from EFruII to fructose.     

Factors Affecting the Synthesis and Degradation of Ribulose-1,5-Diphosphate Carboxylase in Hydrogenomonas facilis and Hydrogenomonas eutropha

Hydrogenomonas facilis and H. eutropha cultured in fructose medium retained high levels of ribulose-1,5-diphosphate carboxylase only when the following conditions were fulfilled: low aeration, FeCl(3) addition to fructose medium, and cell harvest at or prior to mid-exponential phase of growth. Repression of carboxylase synthesis was demonstrated under conditions of high oxygen tension during growth of H. eutropha on fructose. Upon depletion of fructose in the growth medium, carboxylase activity fell abruptly in both organisms. The decline could not be attributed to a repressive mechanism. Rapid inactivation of carboxylase was promoted by transfer of mid-exponential-phase H. eutropha to a basal salts medium lacking fructose. During severe fructose starvation, N(2), H(2), 80% H(2) to 20% air, 2,4-dinitrophenol, actinomycin D, streptomycin, bicarbonate, and magnesium ion deficiency spared carboxylase. Nitrogen starvation or chloramphenicol afforded no protection during severe starvation. In vitro inactivation was also demonstrated in crude cell-free extracts from nonstarved, fructose-grown H. eutropha. Substrate bicarbonate protected against this loss. Inactivation of the carboxylase could not be demonstrated either by starvation of autotrophically grown cells or in autotrophic extracts. Autotrophic extracts mixed with heterotrophic extracts lost their carboxylase activity, but mixing with heterotrophic extracts that had been heated to 50 C resulted in no loss of activity. Mechanisms are proposed to accommodate these observations.     

Oviposition by Lobesia botrana is stimulated by sugars detected by contact chemoreceptors

Abstract.  The influence of glucose, fructose and sucrose on oviposition site selection by Lobesia botrana is studied by combining behavioural and electrophysiological experiments. Oviposition choice assays, using surrogate grapes treated with grape berry surface extracts of Vitis vinifera cv. Merlot at different development stages, show that L. botrana females are most stimulated by extracts of mature berries containing the highest concentrations of glucose and fructose. Choice assays reveal that the oviposition response to these sugars is dose-dependant (with a threshold of the applied solution = 10 m m and a maximum stimulation at 1  m ) and that females are more sensitive to fructose than to glucose. Tarsal contact-chemoreceptor sensilla are unresponsive to stimulation with sugars but the ovipositor sensilla contain at least one neurone most sensitive to fructose and sucrose with a threshold of approximately 0.5 m m . Corresponding to the behavioural data, glucose is significantly less stimulatory to sensilla than fructose or sucrose. It is argued that fructose may be of special importance for herbivorous insects exploiting fruit as an oviposition site.     

Enzymic assay of 10 to 10 moles of sucrose in plant tissues

Procedures are described for measuring sucrose in plant extracts or freeze-dried tissue in the range between 10⁻⁷ and 10⁻¹⁴ moles. The method is based on the destruction of pre-existing glucose and fructose, followed by the hydrolysis of sucrose and reduction of NADP⁺ by a series of coupled enzymic reactions. Depending on the sensitivity required, the NADPH is determined directly with a spectrophotometer or a fluorometer, or is amplified as much as 30,000 times before fluorometric assay. The procedures suggested for the macro level are simpler than current methods, and those suggested for microanalysis are several orders of magnitude more sensitive.     

P-nitrophenyl phosphate as substrate for rabbit liver fructose diphosphatase

Purified rabbit liver fructose diphosphatase has been found to catalyze the hydrolysis of p-nitrophenyl phosphate, PNPP. It has been established that the hydrolysis of p-nitrophenyl phosphate is due to fructose diphosphatase through studies of the chromatographic properties of the enzyme, its temperature sensitivity, dependence on divalent cations and its inhibition by fructose diphosphate. The K_m for PNPP is 6 × 10⁻³M at pH 9.2, 5 × 10⁻⁴M at pH 7.5. This substrate should facilitate studies of the kinetics and mechanism of action of fructose diphosphatase and the comparison of this enzyme with other alkaline phosphatases.     

Effect of fructose 1-phosphate on the activation of liver glycogen synthase.

The activation (dephosphorylation) of glycogen synthase and the inactivation (dephosphorylation) of phosphorylase in rat liver extracts on the administration of fructose were examined. The lag in the conversion of synthase b into a was cancelled, owing to the accumulation of fructose 1-phosphate. A decrease in the rate of dephosphorylation of phosphorylase a was also observed. The latency re-appeared in gel-filtered liver extracts. Similar latency was demonstrated in extracts from glucagon-treated rats. Addition of fructose 1-phosphate to the extract was able to abolish the latency, and the activation of glycogen synthase and the inactivation of phosphorylase occurred simultaneously. Fructose 1-phosphate increased the activity of glycogen synthase b measured in the presence of 0.2-0.4 mM-glucose 6-phosphate. According to kinetic investigations, fructose 1-phosphate increased the affinity of synthase b for its substrate, UDP-glucose. The accumulation of fructose 1-phosphate resulted in glycogen synthesis in the liver by inducing the enzymic activity of glycogen synthase b in the presence of glucose 6-phosphate in vivo and by promoting the activation of glycogen synthase.     

31P and 13C nuclear magnetic resonance studies of metabolic pathways in Pasteurella multocida characterization of a new mannitol-producing metabolic pathway.

Glucose metabolism of Pasteurella multocida was examined in resting cells in vivo using 13C NMR spectroscopy, in cell-free extracts in vitro using 31P NMR spectroscopy and using enzyme assays. The NMR data indicate that glucose is converted by the Embden-Meyerhof and pentose phosphate pathways. The P. multocida fructose 6-phosphate phosphotransferase activity (the key enzyme of the Embden-Meyerhof pathway) was similar to that of Escherichia coli. Nevertheless, and in contrast to that of E. coli, its activity was inhibited by alpha glycerophosphate. This inhibition is consistent with the very low fructose 6-phosphate phosphotransferase activity found in cell-free extracts of P. multocida using a spectrophotometric method. The dominant end products of glucose metabolism were mannitol, acetate and succinate. Under anaerobic conditions, P. multocida was able to constitutively produce mannitol from glucose, mannose, fructose, sucrose, glucose 6-phosphate and fructose 6-phosphate. We propose a new metabolic pathway in P. multocida where fructose 6-phosphate is reduced to mannitol 1-phosphate by fructose 6-phosphate reductase. Mannitol 1-phosphate produced is then converted to mannitol by mannitol 1-phosphatase.     

Construction of a flocculating yeast for fructose production from inulin

Construction of flocculating yeast lacking for fructose utilisation was realised by integration of the FLO1 flocculation gene in the ribosomal DNA of an hexokinase deficient (hxk1, hxk2) Saccharomyces cerevisiae strain (ATCC36859). Simultaneous production of ethanol and fructose was obtained from glucose/fructose mixtures or from hydrolysed Jerusalem artichoke extracts using the transformed yeast in batch fermentations and in a continuous reactor with internal biomass recycle. This allowed the production of 5 g ethanol/L and 48 g sugars/L containing up to 99 % fructose from diluted hydrolysed Jerusalem artichoke extracts containing 60 g sugars/L. © Rapid Science Ltd. 1998