Similarity of activation of yeast phosphofructokinase by AMP and fructose-2,6-bisphosphate

Phosphofructokinase from yeast is effectively activated by AMP and fructose-2,6-bisphosphate by increasing the affinity of the enzyme to fructose-6-phosphate and the maximum activity toward this substrate. The enzyme is activated by AMP and fructose-2, 6-bisphosphate both at high and at low concentrations of ATP. The half maximum stimulation concentrations of AMP and fructose-2, 6-bisphosphate are about 200 microM and 2 microM, respectively. At saturating concentrations of AMP and fructose-2, 6-bisphosphate similar maximum activities were observed in the dependence of enzyme activity on the concentrations of fructose-6-phosphate. The fructose-6-phosphate affinity is more enhanced by fructose-2, 6-bisphosphate than by AMP.     

The mechanism of rat liver fructose-1,6-bisphosphate inhibition by fructose-2,6-bisphosphate

It was found that a decrease in the activating cation (Mg2+) concentration below [A]0.5 causes the disappearance of cooperativity of the fructose 1.6-bisphosphatase substrate binding sites induced by high fructose 2.6-bisphosphate concentrations without any significant alteration in the extent of the enzyme inhibition. Under these conditions, a competitive type of inhibition (with respect to the substrate) is transformed into a non-competitive type with an increase in the fructose 2.6-bisphosphate concentration. The data obtained confirm the viewpoint that fructose 2.6-bisphosphate binds to the enzyme at two distinct sites, a catalytic and an allosteric ones, differing in their affinity for the inhibitor. It is supposed that the interaction between the allosteric fructose 2.6-bisphosphate binding site and the activator site occupied by Mg2+ is necessary for the cooperative response of the enzyme to the substrate.     

Yeast fructose-2,6-bisphosphate 6-phosphatase is encoded by PHO8, the gene for nonspecific repressible alkaline phosphatase.

Yeast fructose-2,6-bisphosphate 6-phosphatase has been purified 7000-fold by heat treatment, poly(ethylene glycol) precipitation, ion-exchange chromatography with Q-Sepharose Fast Flow and Mono Q followed by affinity chromatography with concanavalin-A-Sepharose and gel filtration with Superose 12. The purified dimeric enzyme contains 1.5 mol zinc and 1.3 mol copper/mol subunit. It reacts with fructose 2,6-bisphosphate [Fru(2,6)P2] as well as with p-nitrophenyl phosphate (NpP) showing a pH optimum at pH 6-6.5 with Fru(2,6)P2 [Plankert, U., Purwin, C. & Holzer, H. (1988) FEBS Lett. 239, 69-72] and above pH 9.0 with NpP. The following observations suggest that activity with both substrates depends on the same protein. (a) During 7000-fold purification, the ratio of activity with NpP to that with Fru(2,6)P2 remained constant. (b) The time course of inactivation of enzyme activity in dilute solution at 30 degrees C is similar for both substrates. (c) At increasing temperatures, inactivation of enzyme activity measured with both substrates proceeds at nearly identical rates. (d) Activity with both substrates is found preferentially in the vacuoles. (e) Mutants defective in the nonspecific alkaline phosphatase coded by the PHO8 gene are also defective in Fru(2,6)P2 6-phosphatase activity. (f) A proteinase A mutant, defective in processing and activation of nonspecific alkaline phosphatase coded by the PHO8 gene, also fails to activate Fru(2,6)P2 6-phosphatase.     

Fructose 2,6-bisphosphate activates the cAMP-dependent phosphorylation of yeast fructose-1,6-bisphosphatase in vitro

Fructose-1,6-bisphosphatase purified from Saccharomyces cerevisiae is phosphorylated in vitro by a cAMP-dependent protein kinase. The phosphorylation reaction incorporates 1 mol of phosphate/mol of enzyme and is greatly stimulated by fructose 2,6-bisphosphate. Fructose 2,6-bisphosphate acts upon fructose-1,6-bisphosphatase, not on the protein kinase. The phosphorylation of fructose 1,6-bisphosphatase lowers its activity by about 50%. The characteristics of the phosphorylation reaction in vitro show that this modification is responsible for the inactivation of fructose-1,6-bisphosphatase observed in vivo.     

Quasi-stationary concentrations of fructose-2,6-bisphosphate in the phosphofructokinase-2/fructose-2,6-bisphosphatase cycle

The cooperation of phosphofructokinase-2 and fructose-2,6-bisphosphatase is investigated. Experimentally derived rate laws of the kinase and bisphosphatase activities introduced into the respective differential equations permitted to describe the time evolution of fructose-2,6-bisphosphate to quasi-stationary levels. The two enzyme activities were found to exert strong temperature dependence. The quasi-stationary levels of fructose-2,6-bisphosphate, however, are independent on temperature.     

Fructose 2,6-bisphosphate in yeast

Fructose 2,6-bisphosphate was identified in $\text{Saccharomyces cerevisiae}$ grown on glucose both by its property to be an acid-labile stimulator of 6-phosphofructo 1-kinase and by its ability to be quantitatively converted into fructose 6-phosphate under mild acid conditions. Fructose 2,6-bisphosphate was undetectable in cells grown on non-glucose sources. When glucose was added to the culture, fructose 2,6-bisphosphate was rapidly synthesized, reaching within 1 min concentrations able to cause a profound inhibition of fructose 1,6-bisphosphatase and a great stimulation of 6-phosphofructo 1-kinase.     

Control of photosynthetic sucrose synthesis by fructose-2,6-bisphosphate

The metabolite levels in the mesophyll of leaves of Zea mays L. have been compared with the regulatory properties of the cytosolic fructose-1,6-bisphosphatase from the mesophyll to show how withdrawal of triose phosphate for sucrose synthesis is reconciled with generation of the high concentrations of triose phosphate which are needed to allow intercellular diffusion of carbon during photosynthesis. i) A new technique is presented for measuring the intercellular distribution of metabolites in maize. The bundle-sheath and mesophyll tissues are partially separated by differential homogenization and filtration through nylon nets under liquid nitrogen. ii) considerable gradients of 3-phosphoglycerate, triose phosphate, malate and phosphoenolpyruvate exist between the mesophyll and bundle sheath which would allow intercellular shuttles to be driven by diffusion. These gradients could result from the distribution of electron transport and the Calvin cycle in maize leaves. iii) consequently, the mesophyll contains high concentrations of triose phosphate and fructose-1,6-bisphosphate. iv) Most of the regulator metabolite fructose-2,6-bisphosphate, is present in the mesophyll. v) The cytosolic fructose-1,6-bisphosphatase has a lower substrate affinity than that found for the enzyme from C₃ species, especially in the presence of inhibitors like fructose-2,6-bisphosphate. vi) This lowered affinity for substrate makes it possible to reconcile use of triose phosphate for sucrose synthesis with the maintenance of the high concentration of triose phosphate in the mesophyll needed for operation of photosynthesis in this species.Abbreviations DHAP Dihydroxyacetonephosphate - Fru1,6-bisP fructose-1,6-bisphosphate - Fru2,6bisP fructose-2,6-bisphosphate - PEP(Case) phosphoenolpyruvate (carboxylase) - PGA 3-phosphoglycerate - Rubisco ribulose-1,5-bisphosphate carboxylase     

Inhibition of fructose-1,6-bisphosphatase by fructose 2,6-bisphosphate

Rat liver fructose-1,6-bisphosphatase, which was assayed by measuring the release of 32P from fructose 1,6-[1-32P]bisphosphate at pH 7.5, exhibited hyperbolic kinetics with regard to its substrate. beta-D-Fructose 2,6-bisphosphate, an activator of hepatic phosphofructokinase, was found to be a potent inhibitor of the enzyme. The inhibition was competitive in nature and the Ki was estimated to be 0.5 microM. The Hill coefficient for the reaction was 1.0 in the presence and absence of fructose 2,6-bisphosphate. Fructose 2,6-bisphosphate also enhanced inhibition of the enzyme by the allosteric inhibitor AMP. The possible role of fructose 2,6-bisphosphate in the regulation of substrate cycling at the fructose-1,6-bisphosphatase step is discussed.     

14-3-3s regulate fructose-2,6-bisphosphate levels by binding to PKB-phosphorylated cardiac fructose-2,6-bisphosphate kinase/phosphatase

The cardiac isoform of 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase (PFK-2), regulator of the glycolysis-stimulating fructose-2,6-bisphosphate, was among human HeLa cell proteins that were eluted from a 14-3-3 affinity column using the phosphopeptide ARAApSAPA. Tryptic mass fingerprinting and phospho-specific antibodies showed that Ser466 and Ser483 of 14-3-3-affinity-purified PFK-2 were phosphorylated. 14-3-3 binding was abolished by selectively dephosphorylating Ser483, and 14-3-3 binding was restored when both Ser466 and Ser483 were phosphorylated with PKB, but not when Ser466 alone was phosphorylated by AMPK. Furthermore, the phosphopeptide RNYpS(483)VGS blocked binding of PFK-2 to 14-3-3s. These data indicate that 14-3-3s bind to phosphorylated Ser483. When HeLa cells expressing HA-tagged PFK-2 were co-transfected with active PKB or stimulated with IGF-1, HA-PFK-2 was phosphorylated and bound to 14-3-3s. The response to IGF-1 was abolished by PI 3-kinase inhibitors. In addition, IGF-1 promoted the binding of endogenous PFK-2 to 14-3-3s. When cells were transduced with penetratin-linked AARAApSAPA, we found that this reagent bound specifically to 14-3-3s, blocked the IGF-1-induced binding of HA-PFK-2 to 14-3-3s, and completely inhibited the IGF-1-induced increase in cellular fructose-2,6-bisphosphate. These findings suggest that PKB-dependent binding of 14-3-3s to phospho-Ser483 of cardiac PFK-2 mediates the stimulation of glycolysis by growth factor.     

Arg-257 and Arg-307 of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase bind the C-2 phospho group of fructose-2,6-bisphosphate in the fructose-2,6-bisphosphatase domain.

Rat liver fructose-2,6-bisphosphatase, which catalyzes its reaction via a phosphoenzyme intermediate, is evolutionarily related to the phosphoglycerate mutase enzyme family (Bazan, F., Fletterick, R., and Pilkis, S.J. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9642-9646). Arg-7 and Arg-59 of the yeast phosphoglycerate mutase have been postulated to be substrate-binding residues based on the x-ray crystal structure. The corresponding residues in rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, Arg-257 and Arg-307, were mutated to alanine. The Arg257Ala and Arg307Ala mutants and the wild-type enzyme were expressed in Escherichia coli and then purified to homogeneity. Both mutant enzymes had identical far and near UV circular dichroism spectra and 6-phosphofructo-2-kinase activities when compared with the wild-type enzyme. However, the Arg257Ala and Arg307Ala mutants had altered steady state fructose-2,6-bisphosphatase kinetic properties; the Km values for fructose-2,6-bisphosphate of the Arg257Ala and Arg307Ala mutants were increased by 12,500- and 760-fold, whereas the Ki values for inorganic phosphate were increased 7.4- and 147-fold, respectively, as compared with the wild-type values. However, the Ki values for the other product, fructose-6-phosphate, were unchanged for the mutant enzymes. Although both mutants exhibited parallel changes in kinetic parameters that reflect substrate/product binding, they had opposing effects on their respective maximal velocities; the maximal velocity of Arg257Ala was 11-fold higher, whereas that for Arg307Ala was 700-fold lower, than that of the wild-type enzyme. Pre-steady state kinetic studies demonstrated that the rate of phosphoenzyme formation for Arg307Ala was at least 4000-fold lower than that of the wild-type enzyme, whereas the rate for Arg257Ala was similar to the wild-type enzyme. Furthermore, consistent with the Vmax changes, the rate constant for phosphoenzyme breakdown for Arg257Ala was increased 9-fold, whereas that for Arg307Ala was decreased by a factor of 500-fold, as compared with the wild-type value. The results indicate that both Arg-257 and Arg-307 interact with the reactive C-2 phospho group of fructose 2,6-bisphosphate and that Arg-307 stabilizes this phospho group in the transition state during phosphoenzyme breakdown, whereas Arg-257 stabilizes the phospho group of the ground state phosphoenzyme intermediate.     

Studies on the entry of fructose-2,6-bisphosphate into chloroplasts

The regulatory metabolite fructose-2,6-bisphosphate (Fru-2,6-P₂) has an important function in controlling the intermediary carbon metabolism of leaves. Fru-2,6-P₂ controls two cytosolic enzymes involved in the interconversion of fructose-6-phosphate and fructose-1,6-bisphosphate (fructose-1,6-bisphosphatase and pyrophosphate, fructose-6-phosphate 1-phosphotransferase) and thereby controls the partitioning of photosynthate between sucrose and starch. It has been demonstrated that Fru-2,6-P₂ is present mainly in the cytosol. Here we present evidence that Fru-2,6-P₂ can be taken up by isolated intact chloroplasts but at a very slow rate (about 0.01 micromoles per milligram of chlorophyll per hour). This uptake is time and concentration dependent and is inhibited by PPi. When provided a physiological concentration of Fru-2,6-P₂ (10 micromolar), chloroplasts accumulated up to 0.6 micromolar Fru-2,6-P₂ in the stroma. Elevated plastid Fru-2,6-P₂ levels had no effect on overall photosynthetic rates of isolated chloroplasts. The results indicate that, while Fru-2,6-P₂ enters isolated chloroplasts at a sluggish rate, caution should be exercised in ascribing physiological importance to effects of Fru-2,6-P₂ on chloroplast enzymes.     

Cloning of a second gene encoding 6-phosphofructo-2-kinase in yeast, and characterization of mutant strains without fructose-2,6-bisphosphate

We have identified a new gene, PFK27, that encodes a second inducible 6-phosphofructo-2-kinase in the yeast Saccharomyces cerevisiae. Sequencing shows an open reading frame of 397 amino acids and 45.3 kDa. Amino acid sequence comparisons with other bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoenzymes of various organisms revealed similarities only to the kinase domains. Expression of PFK27 was induced severalfold by glucose and sucrose, but not by galactose or maltose, suggesting that sugar transport might be involved in triggering the induction signal. We have constructed a mutant strain devoid of any fructose-2,6-bisphosphate. The mutant strain grew well on several kinds and concentrations of carbon sources. The levels of hexose phosphates in the cells were increased, but flux rates for glucose utilization and ethanol production were similar to the wild-type strain. However, after the transfer of the mutant cells from respiratory to fermentative growth conditions, growth, glucose consumption and ethanol production were delayed in a transition phase. Our results show that fructose-2,6-bisphosphate is an important effector in vivo of the 6-phosphofructo-1-kinase/fructose-1,6-bisphospha-tase enzyme pair, and is involved in the initiation of glycolysis during the transition to a fermentative mode of metabolism. Nevertheless, it can be effectively replaced by other effectors and regulatory mechanisms during growth on glucose.     

Ethylene-induced increase in fructose-2,6-bisphosphate in plant storage tissues

Treatment of carrot roots with ethylene led to: (a) a doubling of the fructose-2,6-bisphosphate content; (b) a general increase in the concentration of glycolytic intermediates; and (c) an increase in the extractable activity of fructose-6-phosphate,2-kinase, the enzyme synthesizing fructose-2,6-bisphosphate from fructose-6-phosphate and adenosine triphosphate.     

Cyclic AMP,fructose-2,6-bisphosphate and catabolite inactivation of enzymes in the hydrocarbon-assimilating yeast Candida maltosa

The inactivation of fructose-1,6-bisphosphatase, isocitrate lyase and cytoplasmic malate dehydrogenase in Candida maltosa was found to occur after the addition of glucose to starved cells. The concentration of cyclic AMP and fructose-2,6-bisphosphate increased drastically within 30 s when glucose was added to the intact cells of this yeast. From these results it was concluded that catabolite inactivation, with participation of cyclic AMP and fructose-2,6-bisphosphate, is an important control mechanism of the gluconeogenetic sequence in the n-alkane-assimilating yeast Candida maltosa, as described for Saccharomyces cerevisiae.     

The effect of high dose of cortisol on glucose-6-phosphatase and fructose-1,6-bisphosphatase activity,and glucose and fructose-2,6-bisphosphate concentration in carp tissues (Cyprinus carpio L.)

The effect of a high dose of cortisol (200 mg kg(-1) body mass) on juvenile carp was investigated. The activity of glucose-6-phosphatase in liver and of fructose-1,6-bisphosphatase in liver, kidney and muscle, the serum glucose and fructose-2,6-bisphosphate concentration as well as the serum concentration of the injected hormone were measured after 24, 72 and 216 h after intraperitoneal cortisol injection. The activities of fructose-1,6-bisphosphatase in liver and kidney and glucose-6-phosphatase in liver were elevated in comparison with the control, while the fructose-1,6-bisphosphatase activity in the muscle tissue was unchanged. After cortisol injection, the serum glucose level was nearly two times higher after 24 and 72 h and was still 50% higher after 216 h compared with controls. In contrast, the liver fructose-2,6-bisphosphate concentration was unchanged after 24 h. More than two times higher fructose-2,6-bisphosphate concentration was observed in liver after 72 h and it was still elevated after 216 h after the cortisol injection.     

The effect of oxygen on fructose-2, 6-bisphosphate concentration and fructose-6-phosphate, 2-kinase activity in yeast cells

Fructose-2,6-bisphosphate concentration and fructose-6-phosphate,2-kinase activity were measured in yeast cells grown aerobically or anaerobically using glucose as a carbon source. A new improved analytical method using HPLC was employed to measure fructose-2,6-P₂ concentration. Anaerobically-grown yeast cells contain approximately 4-fold higher levels of fructose-2,6-P₂ as compared to aerobically-grown cells in the growth phase of culture. Similarly, fructose-6-P,2-kinase activity is approximately 7-fold higher in the anaerobically-grown cells. These results suggest that the presence of oxygen in the growth medium decreases the content of fructose-2,6-P₂ through inactivation of fructose-6-P,2-kinase.     

Pyruvate kinase from Chlamydia trachomatis is activated by fructose-2,6-bisphosphate

Pyruvate kinase is the final regulatory point in the catabolic Embden-Meyerhoff-Parnas pathway, which controls the carbon flux of glycolytic intermediates and regulates the level of ATP in the cell. In a previous study, we identified, cloned and sequenced pyruvate kinase from the obligate intracellular bacterium Chlamydia trachomatis and demonstrated that the enzyme was active in crude extract. Here, we report the kinetic properties of highly purified C. trachomatis pyruvate kinase. The results indicate that C. trachomatis pyruvate kinase is 53.5 kDa with a pH optima of 7.3. Kinetic studies show that C. trachomatis pyruvate kinase requires both K+ and Mg2+ ions for activity, exhibits sigmoidal kinetics with respect to phosphoenolpyruvate and Michaelis-Menten kinetics with respect to ADP. In addition, C. trachomatis pyruvate kinase is able to use alternative nucleoside diphosphates as phosphate acceptors, although it shows the greatest activity with ADP. In contrast to other bacterial pyruvate kinases that are activated by AMP, our data show that AMP, in addition to ATP and GTP, inhibits C. trachomatis pyruvate kinase. Surprisingly, unlike any other known bacterial pyruvate kinase, C. trachomatis pyruvate kinase was allosterically activated by fructose-2,6-bisphosphate, an important regulatory metabolite that has only been reported in eukaryotes.     

Inhibition of Escherichia coli fructose-1,6-bisphosphatase by fructose 2,6-bisphosphate

Fructose 2,6-bisphosphate, a potent inhibitor of fructose-1,6-bisphosphatases, was found to be an inhibitor of the Escherichia coli enzyme. The substrate saturation curves in the presence of inhibitor were sigmoidal and the inhibition was much stronger at low than at high substrate concentrations. At a substrate concentration of 20 μM, 50% inhibition was observed at 4.8 μM fructose 2,6-bisphosphate. Escherichia coli fructose-1,6-bisphosphatase was inhibited by AMP (K_j = 16 μM) and phosphoenolpyruvate caused release of AMP inhibition. However, neither AMP inhibition nor its release by phosphoenolpyruvate was affected by the presence of fructose 2,6-bisphosphate. The results obtained, together with previous observations, provide further evidence for the fructose 2,6-bisphosphate-fructose-1,6-bisphosphatase active site interaction.     

Lysine 356 is a critical residue for binding the C-6 phospho group of fructose 2,6-bisphosphate to the fructose-2,6-bisphosphatase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

Lysine 356 has been implicated by protein modification studies as a fructose-2,6-bisphosphate binding site residue in the 6-phosphofructo-2-kinase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (Kitajima, S., Thomas, H., and Uyeda, K. (1985) J. Biol. Chem. 260, 13995-14002). However, Lys-356 is found in the fructose-2,6-bisphosphatase domain (Bazan, F., Fletterick, R., and Pilkis, S. J. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9642-9646). In order to ascertain whether Lys-356 is involved in fructose-2,6-bisphosphatase catalysis and/or domain/domain interactions of the bifunctional enzyme, Lys-356 was mutated to Ala, expressed in Escherichia coli, and then purified to homogeneity. Circular dichroism experiments indicated that the secondary structure of the Lys-356-Ala mutant was not significantly different from that of the wild-type enzyme. The Km for fructose 2,6-bisphosphate and the Ki for the noncompetitive inhibitor, fructose 6-phosphate, for the fructose-2,6-bisphosphatase of the Lys-356-Ala mutant were 2700- and 2200-fold higher, respectively, than those of the wild-type enzyme. However, the maximal velocity and the Ki for the competitive product inhibitor, inorganic phosphate, were unchanged compared to the corresponding values of the wild-type enzyme. Furthermore, in contrast to the wild-type enzyme, which exhibits substrate inhibition, there was no inhibition by substrate of the Lys-356-Ala mutant. In the presence of saturating substrate, inorganic phosphate, which acts by relieving fructose-6-phosphate and substrate inhibition, is an activator of the bisphosphatase. The Ka for inorganic phosphate of the Lys-356-Ala mutant was 1300-fold higher than that of the wild-type enzyme. The kinetic properties of the 6-phosphofructo-2-kinase of the Lys-356-Ala mutant were essentially identical with that of the wild-type enzyme. The results demonstrate that: 1) Lys-356 is a critical residue in fructose-2,6-bisphosphatase for binding the 6-phospho group of fructose 6-phosphate/fructose 2,6-bisphosphate; 2) the fructose 6-phosphate binding site is responsible for substrate inhibition; 3) Inorganic phosphate activates fructose-2,6-bisphosphatase by competing with fructose 6-phosphate for the same site; and 4) Lys-356 is not involved in 6-phosphofructo-2-kinase substrate/product binding or catalysis.