Characterization of fructose 1,6-bisphosphatase from bakers' yeast

Active nonphosphorylated fructose bisphosphatase (EC 3.1.3.11) was purified from bakers' yeast. After chromatography on phosphocellulose, the enzyme appeared as a homogeneous protein as deduced from polyacrylamide gel electrophoresis, gel filtration, and isoelectric focusing. A Stokes radius of 44.5 A and molecular weight of 116,000 was calculated from gel filtration. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate resulted in three protein bands of Mr = 57,000, 40,000, and 31,000. Only one band of Mr = 57,000 was observed, when the single band of the enzyme obtained after polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate was eluted and then resubmitted to electrophoresis in the presence of sodium dodecyl sulfate. Amino acid analysis indicated 1030 residues/mol of enzyme including 12 cysteine moieties. The isoelectric point of the enzyme was estimated by gel electrofocusing to be around pH 5.5. The catalytic activity showed a maximum at pH 8.0; the specific activity at the standard pH of 7.0 was 46 units/mg of protein. Fructose 1,6-bisphosphatase b, the less active phosphorylated form of the enzyme, was purified from glucose inactivated yeast. This enzyme exhibited maximal activity at pH greater than or equal to 9.5; the specific activity measured at pH 7.0 was 25 units/mg of protein. The activity ratio, with 10 mM Mg2+ relative to 2 mM Mn2+, was 4.3 and 1.8 for fructose 1,6-bisphosphatase a and fructose 1,6-bisphosphatase b, respectively. Activity of fructose 1,6-bisphosphatase a was 50% inhibited by 0.2 microM fructose 2,6-bisphosphate or 50 microM AMP. Inhibition by fructose 2,6-bisphosphate as well as by AMP decreased with a more alkaline pH in a range between pH 6.5 and 9.0. The inhibition exerted by combinations of the two metabolites at pH 7.0 was synergistic.     

Evidence for different forms of rat liver fructose 1,6-bisphosphatase

Purified liver fructose 1,6-bisphosphatase exhibits different forms upon isoelectric focusing. The enzyme focused at pH 5.75, 5.60, and 5.44. Treatment of the enzyme preparation with the catalytic subunit of cAMP-dependent protein kinase and ATP altered the isoelectric focusing profile such that the bands at 5.75 and 5.60 were diminished, the band at 5.44 increased, and two new bands appeared at 5.30, and 5.18. Fructose 1,6-bisphosphatase may be present in rat liver in different forms, one of which is phosphorylated as the enzyme is isolated.     

Activation of rabbit muscle fructose 1,6-bisphosphatase by histidine and carnosine

Histidine and its derivatives increased rabbit muscle fructose 1,6-bisphosphatase activity at neutral pH with positive cooperativity. In the presence of histidine and carnosine the optimum pH shifted from pH 8.0 to 7.4. The cooperative response of the enzyme to AMP and fructose 1,6-bisphosphate was observed in the presence of the histidine derivatives. Of a number of divalent cations tested, only Zn2+ was found to be an effective inhibitor of enzyme activity at low concentrations. The kinetic data suggested that Zn2+ acted as inhibitor as well as activator for the enzyme activity; a high affinity binding site was associated with Ki of approximately 0.5 microM Zn2+ and a catalytic site was associated with Km of approximately 10 microM Zn2+. Rabbit muscle fructose 1,6-bisphosphatase bound 4 equivalents of Zn2+/mol, presumably 1 per subunit, in the absence of fructose 1,6-bisphosphate. Two equivalents of Zn2+/mol bound to the enzyme were readily removed by dialysis or gel filtration in the absence of a chelating agent. The other two equivalents of Zn2+/mol were removed by histidine and histidine derivatives of naturally occurring chelators with concomitant increase in activity.     

The interaction of fructose 2,6-bisphosphate and AMP with rat hepatic fructose 1,6-bisphosphatase

The binding of the inhibitory ligands fructose 2,6-bisphosphate and AMP to rat liver fructose 1,6-bisphosphatase has been investigated. 4 mol of fructose-2,6-P2 and 4 mol of AMP bind per mol of tetrameric enzyme at pH 7.4. Fructose 2,6-bisphosphate exhibits negative cooperatively as indicated by K'1 greater than K'2 greater than K'3 greater than or equal to K'4 and a Hill plot, the curvature of which indicates K'2/K'1 less than 1, K'3/K'2 less than 1, and K'4/K'3 = 1. AMP binding, on the other hand, exhibits positive cooperativity as indicated by K'1 less than K'2 less than K'3 less than K'4 and an nH of 2.05. Fructose 2,6- and fructose 1,6-bisphosphates enhance the binding of AMP as indicated by an increase in the intrinsic association constants. At pH 9.2, where fructose 2,6-bisphosphate and AMP inhibition of the enzyme are diminished, fructose 2,6-bisphosphate binds with a lower affinity but in a positively cooperative manner, whereas AMP exhibits half-sites reactivity with only 2 mol of AMP bound per mol of tetramer. Ultraviolet difference spectroscopy confirmed the results of these binding studies. The site at which fructose 2,6-bisphosphate binds to fructose 1,6-bisphosphatase has been identified as the catalytic site on the basis of the following. 1) Fructose 2,6-bisphosphate binds with a stoichiometry of 1 mol/mol of monomer; 2) covalent modification of the active site with acetylimidazole inhibits fructose 2,6-bisphosphate binding; and 3) alpha-methyl D-fructofuranoside-1,6-P2 and beta-methyl D-fructofuranoside-1,6-P2, substrate analogs, block fructose 2,6-bisphosphate binding. We propose that fructose 2,6-bisphosphate enhances AMP affinity by binding to the active site of the enzyme and bringing about a conformational change which may be similar to that induced by AMP interaction at the allosteric site.     

The effect of fructose 2,6-bisphosphate on muscle fructose-1,6-bisphosphatase activity

Rat and rabbit muscle fructose 1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) are inhibited by fructose 2,6-bisphosphate. In contrast with the liver isozyme, the inhibition of muscle fructose-1,6-bisphosphatase by fructose 2,6-bisphosphate is not synergistic with that of AMP. Activation of fructose-1,6-bisphosphatase by fructose 2,6-bisphosphate has been observed at high concentrations of substrate. An attempt is made to correlate changes in concentrations of hexose monophosphate, fructose 1,6-bisphosphate and fructose 2,6-bisphosphate with changes in fluxes through 6-phosphofructokinase and fructose-1,6-bisphosphatase in isolated epitrochlearis muscle challenged with insulin and adrenaline.     

The regulation of the interaction between F-actin and muscle fructose 1,6-bisphosphatase

Interaction between rabbit muscle fructose 1,6-bisphosphatase (FBPase) and rabbit muscle F-actin results in heterologous complex formation [A. Gizak, D. Rakus, A. Dzugaj, Histol. Histopathol. 18 (2003) 135]. Calculated on the basis of co-sedimentation-binding experiments and ELISA assay-binding constant (Ka) revealed that FBPase binds to F-actin with Ka equal to 7.4 x 10(4) M(-1). The binding is down-regulated by ligands interacting with the FBPase active site (fructose 6-phosphate, fructose 1,6-bisphosphate, fructose 2,6-bisphosphate) and with the FBPase allosteric inhibitory site (AMP). The binding and the kinetic data suggests that FBPase may bind F-actin using a bipartite motif which includes the amino acids residues involved in the binding of the substrate as well as of the allosteric inhibitor of the enzyme. The in situ co-localization experiment, in which FBPase was diffused into skinned muscle fibres pre-incubated with phalloidin (polymeric actin-interacting toxin), has shown that FBPase binds predominantly to the region of the Z-line.     

Hysteretic behavior of rat liver fructose 1,6-bisphosphatase induced by zinc ions

The measurement of the time dependency of the activity of rat liver fructose 1,6-bisphosphatase shows that the enzyme under certain conditions exhibits kinetic hysteretics. After addition of the substrate, the enzyme is initially in a state characterized by a “high” K_m of about 2 μm. During the reaction the enzyme is converted in a slow process to a low K_m form (K_m is about 0.5 μm). The transition is accompanied by a decrease in V. It is concluded that the hysteretic behavior is caused by binding of the Zn²⁺ substrate complex to the enzyme. The earlier reported effect of glucagon treatment on the activity of fructose 1,6-bisphosphate (O. D. Taunton, F. B. Stifel, H. L. Greene, and R. H. Herman (1974) J. Biol. Chem.249, 7228–7239) was reinvestigated, taking into account the hysteretic behavior. Under conditions where the pyruvate kinase activity is decreased by glucagon injection, no activity change of fructose 1,6-bisphosphatase is observed. It can be suggested that for studies concerning the effects of incubation or hormone treatment on fructose 1,6-bisphosphatase, the complex kinetics of the rat liver enzyme has to be taken into account.     

Interaction between muscle aldolase and muscle fructose 1,6-bisphosphatase results in the substrate channeling

Fructose 1,6-bisphosphatase (FBPase) is known to form a supramolecular complex with alpha-actinin and aldolase on both sides of the Z-line in skeletal muscle cells. It has been proposed that association of aldolase with FBPase not only desensitizes muscle FBPase toward AMP inhibition but it also might enable the channeling of intermediates between the enzymes [Rakus et al. (2003) FEBS Lett. 547, 11-14]. In the present paper, we tested the possibility of fructose 1,6-bisphosphate (F1,6-P(2)) channeling between aldolase and FBPase using the approach in which an inactive form of FBPase competed with active FBPase for binding to aldolase and thus decreased the rate of aldolase-FBPase reaction. The results showed that F1,6-P(2) is transferred directly from aldolase to FBPase without mixing with the bulk phase. Further evidence that F1,6-P(2) is channeled from aldolase to FBPase comes from the experiments investigating the inhibitory effect of a high concentration of magnesium ions on aldolase-FBPase activity. FBPase in a complex with aldolase, contrary to free muscle FBPase, was not inhibited by high Mg(2+) concentrations, which suggests that free F1,6-P(2) was not present in the assay mixture during the reaction. A real-time interaction analysis between aldolase and FBPase revealed a dual role of Mg(2+) in the regulation of the aldolase-FBPase complex stability. A physiological concentration of Mg(2+) increased the affinity of muscle FBPase to muscle aldolase, whereas higher concentrations of the cation decreased the concentration of the complex. We hypothesized that the presence of Mg(2+) stabilizes a positively charged cavity within FBPase and that it might enable an interaction with aldolase. Because magnesium decreased the binding constant (K(a)) between aldolase and FBPase in a manner similar to the decrease of K(a) caused by monovalent cations, it is postulated that electrostatic attraction might be a driving force for the complex formation. It is presumed that the biological relevance of F1,6-P(2) channeling between aldolase and FBPase is protection of this glyconeogenic, as well as glycolytic, intermediate against degradation by cytosolic aldolase, which is one of the most abundant enzyme of glycolysis.     

Nuclear accumulation of fructose 1,6-bisphosphatase is impaired in diabetic rat liver

Using a streptozotocin-induced type 1 diabetic rat model, we analyzed and separated the effects of hyperglycemia and hyperinsulinemia over the in vivo expression and subcellular localization of hepatic fructose 1,6-bisphosphatase (FBPase) in the multicellular context of the liver. Our data showed that FBPase subcellular localization was modulated by the nutritional state in normal but not in diabetic rats. By contrast, the liver zonation was not affected in any condition. In healthy starved rats, FBPase was localized in the cytoplasm of hepatocytes, whereas in healthy re-fed rats it was concentrated in the nucleus and the cell periphery. Interestingly, despite the hyperglycemia, FBPase was unable to accumulate in the nucleus in hepatocytes from streptozotocin-induced diabetic rats, suggesting that insulin is a critical in vivo modulator. This idea was confirmed by exogenous insulin supplementation to diabetic rats, where insulin was able to induce the rapid accumulation of FBPase within the hepatocyte nucleus. Besides, hepatic FBPase was found phosphorylated only in the cytoplasm, suggesting that the phosphorylation state is involved in the nuclear translocation. In conclusion, insulin and not hyperglycemia plays a crucial role in the nuclear accumulation of FBPase in vivo and may be an important regulatory mechanism that could account for the increased endogenous glucose production of liver of diabetic rodents.     

The origin of the high sensitivity of muscle fructose 1,6-bisphosphatase towards AMP

Adenosine 5'-monophosphate (AMP) inhibits muscle fructose 1,6-bisphosphatase (FBPase) about 44 times stronger than the liver isozyme. The key role in strong AMP binding to muscle isozyme play K20, T177 and Q179. Muscle FBPase which has been mutated towards the liver enzyme (K20E/T177M/Q179C) is inhibited by AMP about 26 times weaker than the wild-type muscle enzyme, but it binds the fluorescent AMP analogue, 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-monophosphate (TNP-AMP), similarly to the wild-type liver enzyme. The reverse mutation of liver FBPase towards the muscle isozyme significantly increases the affinity of the mutant to TNP-AMP. High affinity to the inhibitor but low sensitivity to AMP of the liver triple mutant suggest differences between the isozymes in the mechanism of allosteric signal transmission.     

Characterization of a regulatory mutant of fructose 1,6-bisphosphatase in Saccharomyces carlsbergensis.

Summary The fdp mutation has been localized on the genome of Saccharomyces carlsbergensis, on chromosome II, between lys2 and tyr1, at a map distance of 31 centimorgan from lys2.Since the fdp mutant does not grow on glucose, fructose, mannose and sucrose, hexose transport and a number of enzymes of carbon metabolism were tested, but no significant differences could be found between the wild type and the mutant. Only the regulatory properties of glycogen synthetase are changed in the mutant, but it is doubtfull whether this can explain its phenotype.The disorganization of carbon metabolism of the mutant upon addition of glucose to the medium was analyzed in more detail. The most prominent feature observed until now is the accumulation of free glucose and hexose phosphates in the cell. This result indicates that somehow the feedback control between hexose transport and metabolism is impaired. Hexose phosphates are known to be toxic to many cells, including yeast. Therefore, accumulation of hexose phosphates in the presence of glucose in the medium, can explain the absence of growth on this carbon source.     

Evidence for an interaction between fructose 1,6-bisphosphatase and fructose 1,6-bisphosphate aldolase.

Three distinct lines of evidence suggest interaction and possible complex formation between fructose 1,6-biphosphate aldolase (EC 4.1.2.13) and fructose 1,6-biphosphatase (EC 3.1.3.11) from rabbit liver. (1) Fructose 1,6-biphosphatase, which does not contain tryptophan, causes changes in the fluorescence emission spectrum of tryptophan in rabbit liver aldolase. (2) Aldolase reduces the affinity of binding of Zn²⁺ to the two high-affinity sites of fructose 1,6-biphosphatase. (3) Gel penetration coefficients are decreased for both enzymes when they are tested together, as compared to the coefficients observed when each is tested separately. These interactions were not observed when either liver enzyme was replaced by the corresponding enzyme purified from rabbit muscle; this specificity for enzymes purified from the same tissue excludes effects attributable to the catalytic activities of the enzyme. Maximum interaction was observed in the pH range between 8.0 and 8.5 and appeared to require the presence of two fructose 1,6-biphosphatase tetramers per tetramer of aldolase. The change in fluorescence emission spectrum was also observed, to a smaller extent, when muscle fructose 1,6-biphosphatase was added to a solution of muscle aldolase.     

Catabolite inactivation of fructose 1,6-bisphosphatase inKluyveromyces fragilis

Catabolite inactivation of fructose 1,6-bisphosphatase inKluyveromyces fragilis was found to occur as a one-step process with a half-life of approximately 90 min in contrast to the two-step process previously reported forSaccharomyces cerevisiae. No rapid initial 50% loss of activity immediately after a glucose-induced catabolite inactivation was found; nevertheless, fructose 1,6-bisphosphatase was rapidly phosphorylated within 5 min of glucose addition. This result supports the hypothesis that protein phosphorylation serves as a signal for the specific degradation of fructose 1,6-bisphosphatase during catabolite inactivation.     

The interaction of fructose 2,6-bisphosphate with an allosteric site of rat liver fructose 1,6-bisphosphatase

Rat liver fructose 1,6-bisphosphatase can be protected against partial inactivation by N-ethylmaleimide by low concentrations of fructose 2,6-bisphosphate or high concentrations of fructose 1,6-bisphosphate. The partially inactivated enzyme has a much reduced sensitivity to high substrate inhibition and has lost the sigmoid component of the inhibition by fructose 2,6-bisphosphate; this compound is a simple linear competitive inhibitor of the modified enzyme. The results suggest that fructose 2,6-bisphosphate can bind to the enzyme at two distinct sites, the catalytic site and an allosteric site. High levels of fructose 1,6-bisphosphate probably inhibit by binding to the allosteric site.     

Effect of dietary excess-histidine on fructose 1,6-bisphosphatase and 6-phosphofructokinase activities, and activation of fructose 1,6-bisphosphatase by basic amino acids in rat liver.

1. Dietary excess histidine caused an increase in the total activity of fructose 1,6-bisphosphatase, and a decrease in 6-phosphofructokinase in the liver. 2. The hepatic concentrations of free histidine and lysine were higher in rats fed a histidine-excess diet. 3. The addition of histidine, lysine or arginine to the assay mixture for fructose 1,6-bisphosphatase resulted in a significant increase in its activity. The 6-phosphofructokinase activity in the liver was not enhanced by the addition of histidine to the assay mixture.