Identification and characterization of cytosolic fructose-1,6-bisphosphatase in Euglena gracilis

Euglena gracilis has the ability to accumulate a storage polysaccharide, a β-1,3-glucan known as paramylon, under aerobic conditions. Under anaerobic conditions, E. gracilis cells degrade paramylon and synthesize wax esters. Cytosolic fructose-1,6-bisphosphatase (FBPase) appears to be a key enzyme in gluconeogenesis and position branch point of carbon partitioning between paramylon and wax ester biosynthesis. We herein identified and characterized cytosolic FBPase from E. gracilis. The K_m and V_max values of EgFBPaseIII were 16.5 ± 1.6 μM and 30.4 ± 7.2 μmol min^?1 mg protein^?1, respectively. The activity of EgFBPaseIII was not regulated by AMP or reversible redox modulation. No significant differences were observed in the production of paramylon in transiently suppressed EgFBPaseIII gene expression cells by RNAi (KD-EgFBPaseIII); nevertheless, FBPase activity was markedly decreased in KD-EgFBPaseIII cells. On the other hand, the growth of KD-EgFBPaseIII cells was slightly higher than that of control cells.     

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 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.     

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.     

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.     

On the nucleotide binding domain of fructose-1,6-bisphosphatase.

Native chicken liver fructose-1,6-bisphosphatase (Fru-P₂ase) can bind to blue dextranSepharose affinity column and is not displaced by its sugar-phosphate substrate; however; it is readily eluted by the inhibitor 5′-AMP. Treatment of Fru-P₂ase with pyridoxal 5′-phosphate (pyridoxal-P) in the presence of the substrate, fructose 1,6-bisphosphate, followed by reduction with NaBH₄ leads to the formation of active pyridoxal-P derivatives of the enzyme showing diminished sensitivity to AMP inhibitor. The modified enzyme does not bind to the affinity column. On the other hand, in the presence of AMP modification of Fru-P₂ase with pyridoxal-P occurs at the catalytic site; this modification does not alter its binding behavior toward the dye ligand. Blue dextran can also protect Fru-P₂ase against AMP inhibition, and it is a competitive desensitizer for the nucleotide ligand. The results establish that blue dextran binds specifically to the allosteric site of the enzyme, and that the structure of this site may resemble that of the dinucleotide fold in other enzymes. Like native Fru-P₂ase, digestion of pyridoxal-P-Fru-P₂ase (with regulatory properties altered) with subtilisin causes a severalfold increase in the catalytic activity measured at pH 9.2, without significant change in the activity at pH 7.5, and produces a peptide with 56 amino acids. The residual subunit, M_r ~ 30,000, was found to contain all of the incorporated pyridoxal-P.     

Metaphosphate in the active site of fructose-1,6-bisphosphatase

The hydrolysis of a phosphate ester can proceed through an intermediate of metaphosphate (dissociative mechanism) or through a trigonal bipryamidal transition state (associative mechanism). Model systems in solution support the dissociative pathway, whereas most enzymologists favor an associative mechanism for enzyme-catalyzed reactions. Crystals of fructose-1,6-bisphosphatase grow from an equilibrium mixture of substrates and products at near atomic resolution (1.3 A). At neutral pH, products of the reaction (orthophosphate and fructose 6-phosphate) bind to the active site in a manner consistent with an associative reaction pathway; however, in the presence of inhibitory concentrations of K+ (200 mm), or at pH 9.6, metaphosphate and water (or OH-) are in equilibrium with orthophosphate. Furthermore, one of the magnesium cations in the pH 9.6 complex resides in an alternative position, and suggests the possibility of metal cation migration as the 1-phosphoryl group of the substrate undergoes hydrolysis. To the best of our knowledge, the crystal structures reported here represent the first direct observation of metaphosphate in a condensed phase and may provide the structural basis for fundamental changes in the catalytic mechanism of fructose-1,6-bisphosphatase in response to pH and different metal cation activators.     

Allosteric inhibition of fructose-1,6-bisphosphatase by anilinoquinazolines

Anilinoquinazolines currently of interest as inhibitors of tyrosine kinases have been found to be allosteric inhibitors of the enzyme fructose 1,6-bisphosphatase. These represent a new approach to inhibition of F16BPase and serve as leads for further drug design. Enzyme inhibition is achieved by binding at an unidentified allosteric site.     

Reversible denaturation with urea of rabbit liver fructose-1,6-bisphosphatase

Denaturation of fructose-1,6-bisphosphatase (Fru-P2-ase, EC 3.1.3.11) by urea and renaturation of denatured enzyme has been investigated. Denaturation lowers the specific activity of the enzyme but even at 8 M urea concentration in the presence of sucrose the activity of the enzyme is detectable. Centrifugation of the enzyme in a sucrose density gradient at 4 M urea reveals one peak of protein corresponding to a dimer. Denaturation increases intensity of intrinsic fluorescence of Fru-P2-ase and causes a red shift of fluorescence peak of the thioisoindole derivative of the enzyme. Renaturation of the denatured enzyme followed as the reappearance of enzymatic activity in the presence and absence of bovine serum albumin (BSA) is characterised by first order kinetics, k = 1.78 X 10(-3) s-1. The presence of BSA does not affect the rate of renaturation but perceptibly increases the recovery of enzymatic activity. A 100% recovery of Fru-P2-ase activity is observed at 0.5 micrograms/mL concentration of the enzyme and 2 mg/mL of BSA.     

Spontaneous subunit exchange in porcine liver fructose-1,6-bisphosphatase

No evidence to date suggests the possibility of subunit exchange between tetramers of mammalian fructose-1,6-bisphosphatase. An engineered fructose-1,6-bisphosphatase, with subunits of altered electrostatic charge, exhibits spontaneous subunit exchange with wild-type enzyme in the absence of ligands. The exchange process reaches equilibrium in approximately 5 h at 4 degrees C, as monitored by non-denaturing gel electrophoresis and anion exchange chromatography. Active site ligands, such as fructose 6-phosphate, abolish subunit exchange at the level of the monomer, but permit dimer-dimer exchanges. AMP, alone or in the presence of active site ligands, abolishes all exchange processes. Exchange phenomena may play a role in the kinetic mechanism of allosteric regulation of fructose-1,6-bisphosphatase.     

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.     

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 regulatory properties of chloroplast fructose-1,6-bisphosphatase.

The regulatory properties of chloroplast fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, (EC 3.1.3.11) were examined with a homogeneous enzyme preparation isolated from spinach leaves. The activation of the enzyme, that was earlier shown to occur via reduced thioredoxin, was found to be accompanied by a structural change that took place more slowly than the rate of catalysis. The recently found deactivation of the thioredoxin-activated enzyme by physiological oxidants such as oxidized glutathione and dehydroascorbic acid was also slow relative to catalysis. Under the conditions used, the activated enzyme showed a pH optimum of about 8.0, whereas the corresponding value for the non-activated form was pH 8.8. The importance of the thioredoxin-linked mechanism of enzyme regulation that is effected through photoreduced ferredoxin and ferredoxin-thioredoxin reductase is discussed in relation to other light-controlled regulatory agents in chloroplasts.     

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.     

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.     

Amino acid sequence of spinach chloroplast fructose-1,6-bisphosphatase

The amino acid sequence of the spinach chloroplast fructose-1,6-bisphosphatase (FBPase) subunit has been determined. Placement of the 358 residues in the polypeptide chain was based on automated Edman degradation of the intact protein and of peptides obtained by enzymatic or chemical cleavage. The sequence of spinach chloroplast FBPase shows clear homology (ca. 40%) to gluconeogenic (mammalian, yeast, and Escherichia coli) fructose-1,6-bisphosphatases and 80% homology with the wheat chloroplast enzyme. The two chloroplast enzymes show near the middle of the structure a unique sequence insert probably involved in light-dependent regulation of the chloroplast FBPase enzyme activity. This sequence insert contains two cysteines separated by only 4 amino acid residues, a characteristic feature of some enzymes containing redox-active cysteines. The recent X-ray crystallographic resolution of pig kidney FBPase (H. Ke, C. M. Thorpe, B. A. Seaton, F. Marcus, and W. N. Lipscomb, 1989, Proc. Natl. Acad. Sci. USA 86, 1475-1479) has allowed the discussion of the amino acid sequence of spinach chloroplast FBPase in structural terms. It is to be noted that most of pig kidney FBPase residues shown to be either at (or close to) the sugar bisphosphate binding site or located at the negatively charged metal binding pocket are conserved in the chloroplast enzyme. The unique chloroplast FBPase insert presumably involved in light-dependent activation of the enzyme via a thioredoxin-linked mechanism can be accommodated in the surface of the FBPase molecule.