Purification and properties of fructose-1,6-bisphosphatase of Bacillus subtilis.

Fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrase, EC 3.1.3.11) of Bacillus subtilis is a constitutive enzyme that was purified 1000-fold (30% yield) to 80% purity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis where it exhibits a band corresponding to 72,000 daltons. It sediments at 15 S in sucrose density gradients indicating a molecular weight of 380,000, but apparently is very asymmetric. Its activity is irreversibly inactivated in the absence of Mn2+. The enzyme specifically catalyzes dephosphorylation of D-fructose 1,6-bisphosphate with a pH optimum of 8.0. It has 40 to 60% of full activity in the absence of P-enolpyruvate; 20 microM P-enolpyruvate activates it maximally. High concentrations of monovalent cations also activate, NH4+ being most effective. Inhibitors fall into two groups. 1) Nucleoside monophosphates, phosphorylated coenzymes, and polynucleotides inhibit competitively with P-enolpyruvate (AMP (Ki = 2 microM) and dAMP are most effective). 2) The inhibition by nucleoside di- and triphosphates, PPi, and highly phosphorylated nucleotides (guanosine 5'-triphosphate 3'-diphosphate (pppGpp) and adenosine 5'-triphosphate 3'-diphosphate are most effective) is not competed by P-enolpyruvate but is partially overcome by fructose 1,6-bisphosphate (2 microM). Therefore, highly phosphorylated nucleotides (pppGpp and others), produced in over 0.2 mM concentrations upon step down from fast to slow growth rates (Gallant, J., and Lazzarini, R.A. (1976) in Protein Synthesis (McConkey, E.H., ed) Vol. 2, pp. 309-349, Marcel Dekker, Inc., New York), can reduce the conversion rate of fructose 1,6-bisphosphate to fructose 6-phosphate during gluconeogenesis. Comparing glycolytic growth on D-glucose and gluconeogenic growth on L-malate, the intracellular concentrations of fructose 1,6-bisphosphate differ but are both above the Km (13 microM) of the enzyme, those of AMP are similar, whereas those of P-enolpyruvate (0.18 mM versus 1.3 mM) indicate that the enzyme has only 40% of its full activity during glycolysis; nucleotides other than AMP may inhibit additionally. Thus, the futile cycle of fructose 1,6-bisphosphate synthesis and degradation during glycolysis is partially avoided, but the cells are poised for rapid adaptation upon change to gluconeogenic growth conditions.     

Purification and characterization of fructose-1,6-bisphosphatase from bovine brain

Fructose-1,6-bisphosphatase from bovine brain tissue has been purified to near homogeneity. This enzyme is similar to other mammalian fructose-1,6-bisphosphatases in many respects, and its properties are distinctly different from those reported for the enzyme from rat brain [A. L. Majumder and F. Eisenberg (1977) Proc. Natl. Acad. Sci. USA 74, 3222-3225; S. Chattoraj and A. L. Majumder (1986) Biochem. Biophys. Res. Commun. 139, 571-580]. The bovine enzyme (sp act 4, pH ratio (7.5/9.6) = 3.6) has a pH optimum of 7.5. The Km is 2 microM. Divalent metal ion is required for activity, and Vmax is obtained at either 4 mM Mg2+ or 0.3 mM Mn2+. Fructose 2,6-bisphosphate is a competitive inhibitor (Ki = 0.07 microM), and AMP a noncompetitive inhibitor (kis = 24 microM, Kii = 10 microM) of bovine brain fructose-1,6-bisphosphatase. The enzyme activity is enhanced by small amounts of EDTA relative to metal, and AMP inhibits fructose-1,6-bisphosphatase in either the presence or absence of the metal chelator; however, AMP is more effective in the absence of EDTA.     

Purification of human liver fructose-1,6-bisphosphatase

Human liver fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) has been purified 1200-fold using a heat treatment step followed by absorption on phosphocellulose at pH 8 and specific elution with buffer containing the substrate (fructose 1,6-bisphosphate) and allosteric effector (AMP). The enzyme is homogeneous in electrophoresis in polyacrylamide gel, in the presence and absence of denaturing agent. It has a molecular weight of 144 000 and is composed of four identical or nearly identical subunits. Fluorescence spectra indicate that the enzyme does not contain tryptophan residues. The pH optimum is 7.5 and the Km is determined as 0.8 microM. The enzyme is inhibited by AMP in cooperative manner with a K0 x 5 of 6 microM.     

Purification and properties of spinach leaf cytoplasmic fructose-1,6-bisphosphatase.

Cytoplasmic fructose-1,6-bisphosphatase has been purified from spinach leaves to apparent homogeneity. The enzyme is a tetramer of molecular weight about 130,000. At pH 7.5, the Km for fructose 1.6-bisphosphate was 2.5 micron, and for MgCl2 0.13 mM; the enzyme was specific for fructose 1,6-bisphosphate. Saturation with Mg2+ was achieved with lower concentrations at pH 8 than at pH 7. AMP and high concentrations of fructose 1,6-bisphosphate inhibited enzyme activity. Ammonium sulfate relieved the latter inhibition but was itself inhibitory when substrate concentrations were low. Acetylation studies demonstrated that the AMP regulatory site was distinct from the catalytic site. Cytoplasmic fructose-1,6-bisphosphatase may contribute to the regulation of sucrose biosynthesis in plant leaves.     

Studies on the regulation of chloroplast fructose-1,6-bisphosphatase. Activation by fructose 1,6-bisphosphate

Chloroplast fructose-1,6-bisphosphatase (D-fructose 1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) isolated from spinach leaves, was activated by preincubation with fructose 1,6-bisphosphate. The rate of activation was slower than the rate of catalysis, and dependent upon the temperature and the concentration of fructose 1,6-bisphosphate. The addition of other sugar diphosphates, sugar monophosphates or intermediates of the reductive pentose phosphate cycle neither replaced fructose 1,6-bisphosphate nor modified the activation process. Upon activation with the effector the enzyme was less sensitive to trypsin digestion and insensitive to mercurials. The activity of chloroplast fructose-1,6-bisphosphatase, preincubated with fructose 1,6-bisphosphate, returned to its basal activity after the concentration of the effector was lowered in the preincubation mixture. The results provide evidence that fructose-1,6-bisphosphatase resembles other regulatory enzymes involved in photosynthetic CO2 assimilation in its activation by chloroplast metabolites.     

Purification of mRNA coding for rat-liver fructose-1,6-bisphosphatase by polysome immunoabsorption

The mRNA coding for rat liver fructose-1,6-bisphosphatase, which represents approx. 0.46% of total hepatic mRNA, has been purified to near homogeneity. Polysomes from rat liver were allowed to react with antibodies to rabbit anti-fructose-1,6-bisphosphatase purified by affinity chromatography. The complex was immobilized on a protein A-Sepharose column. After the removal of unabsorbed polysomes, the specific mRNA was eluted and chromatographed on an oligo(dT)-cellulose column. This method gave a 183-fold enrichment of the fructose-1,6-bisphosphatase mRNA to greater than 80% homogeneity as determined by electrophoreses of immunoprecipitated in vitro translation products on polyacrylamide slab gels in the presence of sodium dodecyl sulphate.     

Essential arginyl residues in fructose-1,6-bisphosphatase.

Modification of pig kidney fructose-1,6-bisphosphatase with 2,3-butanedione in borate buffer (pH 7.8) leads to the loss of the activation of the enzyme by monovalent cations, as well as to the loss of allosteric adenosine 5'-monophosphate (AMP) inhibition. In agreement with the results obtained for the butanedione modification of arginyl residues in other enzymes, the effects of modification can be reversed upon removal of excess butanedione and borate. Significant protection to the loss of K+ activation was afforded by the presence of the substrate fructose 1,6-bisphosphate, whereas AMP preferentially protected against the loss of AMP inhibition. The combination of both fructose 1,6-bisphosphate and AMP fully protected against the changes in enzyme properties on butanedione treatment. Under the latter conditions, one arginyl residue per mole of enzyme subunit was modified, whereas three arginyl residues were modified by butanedione under conditions leading to the loss of both potassium activation and AMP inhibition. Thus, the modification of two arginyl residues per subunit would appear to be responsible for the change in enzyme properties. The present results, as well as those of a previous report on the subject (Marcus, F. (1975), Biochemistry 14, 3916-3921) support the conclusion that one arginyl residue per subunit is essential for monovalent cation activation, and another arginyl residue is essential for AMP inhibition. A likely role of the latter residue could be its involvement in the binding of the phosphate group of AMP.     

AMP inhibition of pig kidney fructose-1,6-bisphosphatase.

Lys-112 and Tyr-113 in pig kidney fructose-1,6-bisphosphatase (FBPase) make direct interactions with AMP in the allosteric binding site. Both residues interact with the phosphate moiety of AMP while Tyr-113 also interacts with the 3'-hydroxyl of the ribose ring. The role of these two residues in AMP binding and allosteric inhibition was investigated. Site-specific mutagenesis was used to convert Lys-112 to glutamine (K112Q) and Tyr-113 to phenylalanine (Y113F). These amino acid substitutions result in small alterations in k(cat) and increases in K(m). However, both the K112Q and Y113F enzymes show alterations in Mg(2+) affinity and dramatic reductions in AMP affinity. For both mutant enzymes, the AMP concentration required to reduced the enzyme activity by one-half, [AMP](0.5), was increased more than a 1000-fold as compared to the wild-type enzyme. The K112Q enzyme also showed a 10-fold reduction in affinity for Mg(2+). Although the allosteric site is approximately 28 A from the metal binding sites, which comprise part of the active site, these site-specific mutations in the AMP site influence metal binding and suggest a direct connection between the allosteric and the active sites.     

Kinetic studies on the mechanism and regulation of rabbit liver fructose-1,6-bisphosphatase

The interaction of Mg2+, AMP, and fructose 2,6-bisphosphate with respect to rabbit liver fructose-1,6-bisphosphatase was investigated by studying initial-rate kinetics of the system at pH 9.5. A rapid-equilibrium Random Bi Bi mechanism is suggested for the rabbit liver enzyme from the kinetic data. Our kinetic findings indicate that Mg2+ and the inhibitor AMP are mutually exclusive in their binding to fructose-1,6-bisphosphatase. This probably is the mechanism for AMP regulation of fructose-1,6-bisphosphatase and thus, to some extent, gluconeogenesis. A kinetic model for the interaction of these ligands with respect to rabbit liver fructose-1,6-bisphosphatase is presented.     

Kinetic studies of bovine liver fructose-1,6-bisphosphatase.

Initial rate kinetic studies with bovine liver fructose-1,6-bisphosphatase were carried out in both directions of the reaction to determine the sequence of product release from the enzyme. Product inhibition by fructose-6-P was found to be S-linear, I-linear noncompetitive relative to fructose-1,6-bisphosphate, whereas inorganic orthophosphate was determined to be linear competitive with respect to the substrate. The kinetics of the reverse reaction were studied by coupling the phosphatase reaction to the aldolase, triosephosphate isomerase, and glycerolphosphate dehydrogenase reactions. The kinetic results were found to be in harmony with the Uni Bi ordered and random sequential mechanisms as well as a Uni Bi ping-pong mechanism. The nomenclature is that of Cleland (Cleland, W.W. (1963) Biochim. Biophys. Acta 67, 104-137). However, nonkinetic considerations, when taken together with the kinetic results, suggest that the steady state ordered Uni Bi mechanism is the most likely possibility. There is evidence that isomerization of the binary complex of enzyme and phosphate occurs in the kinetic mechanism. Although magnesium is required for the reverse reaction, there is no evidence to suggest that the enzyme discriminates between the magnesium-associated or divalent cation-free forms of the substrates.     

Inactivation of fructose-1,6-bisphosphatase by o-phthalaldehyde

Rabbit liver fructose-1,6-bisphosphatase, a tetramer of identical subunits was rapidly and irreversibly inactivated by o-phthalaldehyde at 25 degrees C (pH 7.3). The second-order rate constant for the inactivation was 30 M-1s-1. Fructose-1,6-bisphosphatase was completely protected from inactivation by the substrate--fructose-1,6-diphosphate but not by the allosteric effector--adenosine monophosphate. The absorption spectrum (lambda max 337 nm) and, fluorescence excitation (lambda max 360 nm) and fluorescence emission spectra (lambda max 405 nm) were consistent with the formation of an isoindole derivative in the subunit between a cysteine and a lysine residue about 3A apart. About 4 isoindole groups per mol of the bisphosphatase were formed following complete loss of the phosphatase activity. This suggests that the amino acid residues of the biphosphatase participating in reaction with o-phthalaldehyde more likely reside at or near the active site instead of allosteric site. The molar transition energy of fructose-1,6-bisphosphatase--o-phthalaldehyde adduct was estimated 121 kJ/mol and compares favorably with 127 kJ/mol for the synthetic isoindole, 1-[(beta-hydroxyethyl)thio]-2-(beta-hydroxyethyl) isoindole in hexane. It is, thus, concluded that the cysteine and lysine residues participating in isoindole formation in reaction between fructose-1,6-bisphosphatase and o-phthalaldehyde are located in a hydrophobic environment.     

Rapid-quench and isotope-trapping studies on fructose-1,6-bisphosphatase

Rapid-quench kinetic measurements yielded presteady-state rate data for rabbit liver fructose-1,6-bisphosphatase (FBPase) (a tetramer of four identical subunits) that are triphasic: the rapid release of Pi (complete within 5 ms), followed by a second reaction phase liberating additional Pi that completes the initial turnover of two or four subunits of the enzyme (requiring 100-150 ms), and a steady-state rate whose magnitude depends on the [alpha-Fru-1,6-P2]/[FBPase] ratio. With Mg2+ in the presence of excess alpha-fructose 1,6-bisphosphate (alpha-Fru-1,6-P2) all four subunits turn over in the pre steady state; with Mn2+ only two of the four are active. Thus the expression of half-site reactivity is a consequence of the nature of the metal ion and not a subunit asymmetry. In the presence of limiting alpha-anomer concentrations only two of the four subunits now remain active with Mg2+ as well as with Mn2+ in the pre steady state. However, so that the amount of Pi released can be accounted for, a beta leads to alpha anomerization or direct beta utilization is required at the active site of one subunit. Such behavior is consistent with the two-state conformational hysteresis displayed by the enzyme and altered affinities manifested within these states for alpha and beta substrate analogues. Under these limiting conditions the subsequent steady-state rate is limited by the beta leads to alpha solution anomerization. These data in combination with pulse--chase experiments permit evaluation of the internal equilibrium, which in the case of Mg2+ is unequivocally higher in favor of product complexes and represents a departure from balanced internal substrate-product complexes.     

Phosphorylated fructose-1,6-bisphosphatase dephosphorylating protein phosphatase from Saccharomyces cerevisiae

Phosphorylation of fructose-1,6-bisphosphatase with cyclic AMP-dependent protein kinase from yeast is accompanied by a 50% decrease in the catalytic activity (Pohlig, G. and Holzer, H. (1985) J. Biol. Chem. 260, 13818-13823). Using reactivation of phoshorylated fructose-1,6-bisphosphatase as assay, a protein phosphatase was about 2,000-fold purified to electrophoretic homogeneity from Saccharomyces cerevisiae. Upon incubation with phosphorylated fructose-1,6-bisphosphatase the purified protein phosphatase not only reverses the 50% inactivation caused by phosphorylation, but also the previously observed change in the pH optimum and in the ratio of activity with Mg2+ or Mn2+. The phosphatase is strongly inhibited by heparin and fluoride. L-Carnitine, orthophosphate, pyrophosphate, and succinate inhibit to 50% at concentrations from 1 to 10 mM. The molecular mass of the native phosphatase was found to be 180,000 Da. Sodium dodecyl sulfate-gel electrophoresis suggested four subunits with a molecular mass of 45,000 Da each. Half-maximal activity was observed with 5 mM Mg2+ or Mn2+, the pH optimum of activity was found at pH 7. Using polyclonal antibodies, disappearance of 32P-labeled fructose-1,6-bisphosphatase and concomitant liberation of the expected amount of inorganic [32P] phosphate was demonstrated.     

A thioredoxin-activated fructose-1,6-bisphosphatase from the cyanobacterium Synechococcus 6301

Fructose-1,6-bisphosphatase was isolated from the cyanobacterium Synechococcus 6301 by acid precipitation, ammonium-sulfate fractionation, and Sephadex gel chromatography. The purified enzyme needed thiols and MgCl₂ for activity. The following K_m-values were obtained: a) for fructose-1,6-bisphosphate: 1.7 mM; b) for MgCl₂: 12.5 mM; c) for dithiocrythritol: 0,56 mM; d) for glutathione: 14 mM; e) for mercaptoethanol: 22 mM; f) for cysteine: 50 mM. Thioredoxin B isolated from this organism will activate this fructose-1,6-bisphosphatase. The K_m of thioredoxin B for this fructose-1,6-bisphosphatase was determined to be 1.7 M, endicotiy that thioredoxin might activate the fructose-1,6-bisphosphatase in Synechococcus in vivo.     

Purification,kinetic studies,and homology model of Escherichia coli fructose-1,6-bisphosphatase

Previous kinetic characterization of Escherichia coli fructose 1,6-bisphosphatase (FBPase) was performed on enzyme with an estimated purity of only 50%. Contradictory kinetic properties of the partially purified E. coli FBPase have been reported in regard to AMP cooperativity and inactivation by fructose-2,6-bisphosphate. In this investigation, a new purification for E. coli FBPase has been devised yielding enzyme with purity levels as high as 98%. This highly purified E. coli FBPase was characterized and the data compared to that for the pig kidney enzyme. Also, a homology model was created based upon the known three-dimensional structure of the pig kidney enzyme. The k_cat of the E. coli FBPase was 14.6 s⁻¹ as compared to 21 s⁻¹ for the pig kidney enzyme, while the K_m of the E. coli enzyme was approximately 10-fold higher than that of the pig kidney enzyme. The concentration of Mg²⁺ required to bring E. coli FBPase to half maximal activity was estimated to be 0.62 mM Mg²⁺, which is twice that required for the pig kidney enzyme. Unlike the pig kidney enzyme, the Mg²⁺ activation of the E. coli FBPase is not cooperative. AMP inhibition of mammalian FBPases is cooperative with a Hill coefficient of 2; however, the E. coli FBPase displays no cooperativity. Although cooperativity is not observed, the E. coli and pig kidney enzymes show similar AMP affinity. The quaternary structure of the E. coli enzyme is tetrameric, although higher molecular mass aggregates were also observed. The homology model of the E. coli enzyme indicated slight variations in the ligand-binding pockets compared to the pig kidney enzyme. The homology model of the E. coli enzyme also identified significant changes in the interfaces between the subunits, indicating possible changes in the path of communication of the allosteric signal.     

Regulation of fructose-1,6-bisphosphatase activity in leaves

Fructose-1,6-bisphosphatase (EC 3.1.3.11) activity increased markedly (greater than 10-fold) upon illumination of wheat leaves. Darkening caused a relatively slow but complete reversal of light activation. The effects of O₂ and CO₂ concentration and light intensity on fructose-bisphosphatase activation were measured. In ratelimiting light, 2% O₂ stimulated enzyme activity, whereas varying the CO₂ concentration had little effect. In saturating light, lowering the oxygen tension had no effect, but CO₂ at near-saturating concentrations for photosynthesis inhibited enzyme activity. Dark inactivation of the enzyme was completely prevented by incubation of leaves in N₂, but was facilitated by O₂, indicating that O₂ is the major oxidant in darkened leaves. It is argued that while fructose bisphosphatase is redox-regulated in leaves, modulation of enzyme activity by this mechanism is unlikely to contribute to the regulation of CO₂ fixation in leaves.     

Chloroplast fructose-1,6-bisphosphatase: structure and function

Redox regulation of photosynthetic enzymes has been a preferred research topic in recent years. In this area chloroplast fructose-1,6-bisphosphatase is probably the most extensively studied target enzyme of the CO₂ assimilation pathway. This review analyzes the structure, biosynthesis, phylogeny, action mechanism, regulation and kinetics of fructose-1,6-bisphosphatase in the light of recent findings on structure–function relationship, and from a molecular biology viewpoint. This revised version was published online in June 2006 with corrections to the Cover Date.