Conditions Required for the Rapid Activation In Vitro of the Chloroplast Fructose-1,6-bisphosphatase

Conditions required for the reductive activation of purified, spinach chloroplast fructose-1,6-bisphosphatase (EC 3.1.3.11) have been determined in vitro. Full reductive activation was observed only when fructose-1,6-bisphosphate and Mg²⁺ were present at the same time as the reducing agent (dithiothreitol). Reduction in the absence either of fructose-1,6-bisphosphate or of Mg²⁺ slowly and irreversibly inactivated the enzyme. The concentration of fructose-1,6-bisphosphate that must be present during reduction for maximum activation depends upon the divalent cation present: it is highest with Mg²⁺, lower with Ca²⁺, and lowest when both Mg²⁺ and Ca²⁺ are present. A scheme for the reductive activation and inactivation of the enzyme is presented.     

Stromal free calcium concentration and light-mediated activation of chloroplast fructose-1,6-bisphosphatase

Light-mediated activation of fructose-1,6-bisphosphatase (EC 3.1.3.11) in intact spinach chloroplasts (Spinacia oleracea L.) is enhanced in the presence of 10⁻⁵ molar external free Ca²⁺. The most pronounced effect is observed during the first minutes of illumination. Ruthenium red, an inhibitor of light-induced Ca²⁺ influx, inhibits this Ca²⁺ stimulated activation. In isolated stromal preparations, the activation of fructose-1,6-bisphosphatase is already enhanced by 2 minutes of exposure to elevated Ca²⁺ concentrations in the presence of physiological concentrations of Mg²⁺ and fructose-1,6-bisphosphate. Maximal activation of the enzyme is achieved between 0.34 and 0.51 millimolar Ca²⁺. The Ca²⁺ mediated activation decreases with increasing fructose-1,6-bisphosphate concentration and with increasing pH. The data are consistent with the proposal that the illumination of chloroplasts leads to a transient increase of free stromal Ca²⁺. In dark-kept chloroplasts the steady-state concentration of free stromal Ca²⁺ is 2.4 to 6.3 micromolar as determined by null point titration. These observations support our previous proposal that light-induced Ca²⁺ influx into chloroplasts does not only influence the cytosolic concentration of free Ca²⁺ but also regulates enzymatic processes inside the chloroplast.     

Control of CO2 fixation regulation of stromal fructose-1,6-bisphosphatase in spinach by pH and Mg2+ concentration

The effect of pH and of Mg²⁺ concentration on the light activated form of stromal fructose-1,6-bisphosphatase (FBPase) was studied using the enzyme rapidly extracted from illuminated spinach chloroplasts. The (fructose-1,6-bisphosphate^4-)(Mg²⁺) complex has been identified as the substrate of the enzyme. Therefore, changes of pH and Mg²⁺ concentrations have an immediate effect on the activity of FBPase by shifting the pH and Mg²⁺ dependent equilibrium concentration of the substrate. In addition, changes of pH and Mg²⁺ concentration in the assay medium have a delayed effect on FBPase activity. A correlation of the activities observed using different pH and Mg²⁺ concentrations indicates, that the effect is not a consequence of the pH and Mg²⁺ concentration as such, but is caused by a shift in the equilibrium concentration of a hypothetical inhibitor fructose-1,6-bisphosphate^3- (uncomplexed), resulting in a change of the activation state of the enzyme. The interplay between a rapid effect on the concentration of the substrate and a delayed effect on the activation state enables a rigid control of stromal FBPase by stromal Mg²⁺ concentrations and pH. Fructose-1,6-bisphosphatase is allosterically inhibited by fructose-6-phosphate in a sigmoidal fashion, allowing a fine control of the enzyme by its product.Abbreviations Fru1,6 bis P fructose-1,6-bisphosphate - Fru6P fructose-6-phosphate - FBPase fructose-1,6-bisphosphatase Some of these results have been included in a preliminary report (Heldt et al. 1984)     

High level expression in Escherichia coli,purification and properties of chloroplast fructose-1,6-bisphosphatase from rapeseed (Brassica napus) leaves

In chloroplasts, the light-modulated fructose-1,6-bisphosphatase catalyzes the formation of fructose 6-bisphosphate for the photosynthetic assimilation of CO₂ and the biosynthesis of starch. We report here the construction of a plasmid for the production of chloroplast fructose-1,6-bisphosphatase in a bacterial system and the subsequent purification to homogeneity of the genetically engineered enzyme. To this end, a DNA sequence that coded for chloroplast fructose-1,6-bisphosphatase of rapeseed (Brassica napus) leaves was successively amplified by PCR, ligated into the Ndel/EcoRI restriction site of the expression vector pET22b, and introduced into Escherichia coli cells. When gene expression was induced by isopropyl--d-thiogalactopyranoside, supernatants of cell lysates were extremely active in the hydrolysis of fructose 1,6-bisphosphate. Partitioning bacterial soluble proteins by ammonium sulfate followed by anion exchange chromatography yielded 10 mg of homogeneous enzyme per 1 of culture. Congruent with a preparation devoid of contaminating proteins, the Edman degradation evinced an unique N-terminal amino acid sequence [A-V-A-A-D-A-T-A-E-T-K-P-]. Gel filtration experiments and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the (recombinant) rapeseed chloroplast fructose-1,6-bisphosphatases was a tetramer [160 kDa] comprised of four identical subunits. Like other chloroplast fructose-1,6-bisphosphatases, the recombinant enzyme was inactive at 1 mM fructose 1,6-bisphosphate and 1 mM Mg²⁺ but became fully active after an incubation in the presence of either 10 mM dithiothreitol or 1 mM dithiothreitol and chloroplast thioredoxin. However, at variance with counterparts isolated from higher plant leaves, the low activity observed in absence of reductants was not greatly enhanced by high concentrations of fructose 1,6-bisphosphate (3 mM) and Mg²⁺ (10 mM). In the catalytic process, all chloroplast fructose-1,6-bisphosphatases had identical features; viz., the requirement of Mg²⁺ as cofactor and the inhibition by Ca²⁺. Thus, the procedure described here should prove useful for the structural and kinetic analysis of rapeseed chloroplast fructose-1,6-bisphosphatase in view that this enzyme was not isolated from leaves.Abbreviation DTT dithiothreitol - PCR polymerase chain reaction - EDTA (ethylenedinitrilo)tetraacetic     

Ostrich fructose-1,6-bisphosphatase: Kinetic characterization of the liver isoenzyme

• 1.1. Purified ostrich (Struthio camelus) liver fructose-1,6-bisphosphatase exhibited an absolute requirement for Mg²⁺.
• 2.2. The enzyme catalyzed the hydrolysis of fructose-1,6-bisphosphate, sedoheptulose-l,7-bisphosphate and ribulose-l,5-bisphosphate.
• 3.3. S_0.5 for substrate was 1.4 μM.
• 4.4. AMP was a potent non-competitive inhibitor with respect to substrate (K_i of 25 μM).
• 5.5. Fructose-2,6-bisphosphate was a potent competitive inhibitor of the enzyme (K_i of 4.8 μM).

     

Fructose-1,6-bisphosphatase from<Emphasis Type="Italic"> Corynebacterium glutamicum</Emphasis>: expression and deletion of the<Emphasis Type="Italic"> fbp</Emphasis> gene and biochemical characterization of the enzyme

The class II fructose-1,6-bisphosphatase gene of Corynebacterium glutamicum, fbp, was cloned and expressed with a N-terminal His-tag in Escherichia coli. Purified, His-tagged fructose-1,6-bisphosphatase from C. glutamicum was shown to be tetrameric, with a molecular mass of about 140 kDa for the homotetramer. The enzyme displayed Michaelis-Menten kinetics for the substrate fructose 1,6-bisphosphate with a K_m value of about 14 µM and a V_max of about 5.4 µmol min^–1 mg^–1 and k_cat of about 3.2 s^–1. Fructose-1,6-bisphosphatase activity was dependent on the divalent cations Mg²⁺ or Mn²⁺ and was inhibited by the monovalent cation Li⁺ with an inhibition constant of 140 µM. Fructose 6-phosphate, glycerol 3-phosphate, ribulose 1,5-bisphosphate and myo-inositol-monophosphate were not significant substrates of fructose-1,6-bisphosphatase from C. glutamicum. The enzymatic activity was inhibited by AMP and phosphoenolpyruvate and to a lesser extent by phosphate, fructose 6-phosphate, fructose 2,6-bisphosphate, and UDP. Fructose-1,6-bisphosphatase activities and protein levels varied little with respect to the carbon source. Deletion of the chromosomal fbp gene led to the absence of any detectable fructose-1,6-bisphosphatase activity in crude extracts of C. glutamicum WTfbp and to an inability of this strain to grow on the carbon sources acetate, citrate, glutamate, and lactate. Thus, fbp is essential for growth on gluconeogenic carbon sources and likely codes for the only fructose-1,6-bisphosphatase in C. glutamicum.     

Purification and properties of fructose 1,6-bisphosphatase from turkey liver.

Fructose 1,6-bisphosphatase (EC 3.1.3.11) has been purified 360-fold from turkey liver. The purified enzyme appears to be homogeneous by disc gel electrophoresis and has a pH profile indistinguishable from that of the enzyme in crude extracts. Mn²⁺ is significantly more effective than Mg²⁺ as the essential metal cofactor of this enzyme. The maximal effect of histidine is equivalent to that of EDTA except that EDTA is more efficient at lower concentrations. The histidine effect is decreased with an increase in pH or if substrate is first bound to the enzyme. The enzyme activity is activated equally by d- and l-forms of histidine. Enzyme affinity for the substrate decreases with an increase in pH. The inhibition by high substrate concentrations observed at pH 7.5 is markedly reduced in the absence of chelating activator or when Mg² is replaced by Mn²⁺ as the metal cofactor. Turkeys liver fructose 1,6-bisphosphatase resembles the enzyme from mammalian sources in that the sensitivity to AMP inhibition is decreased with the increase in pH, temperature, and Mg² concentration.     

Characterization of Hyperthermostable Fructose-1,6-Bisphosphatase from <Emphasis Type="Italic">Thermococcus onnurineus</Emphasis> NA1

To understand the physiological functions of thermostable fructose-1,6-bisphosphatase (TNA1-Fbp) from Thermococcus onnurineus NA1, its recombinant enzyme was overexpressed in Escherichia coli, purified, and the enzymatic properties were characterized. The enzyme showed maximal activity for fructose-1,6-bisphosphate at 95°C and pH 8.0 with a half-life (t _1/2) of about 8 h. TNA1-Fbp had broad substrate specificities for fructose-1,6-bisphosphate and its analogues including fructose-1-phosphate, glucose-1-phosphate, and phosphoenolpyruvate. In addition, its enzyme activity was increased five-fold by addition of 1 mM Mg²⁺, while Li⁺ did not enhance enzymatic activity. TNA1-Fbp activity was inhibited by ATP, ADP, and phosphoenolpyruvate, but AMP up to 100 mM did not have any effect. TNA1-Fbp is currently defined as a class V fructose-1,6-bisphosphatase (FBPase) because it is very similar to FBPase of Thermococcus kodakaraensis KOD1 based on sequence homology. However, this enzyme shows a different range of substrate specificities. These results suggest that TNA1-Fbp can establish new criterion for class V FBPases.     

Studies on the hysteretic properties of chloroplast fructose-1,6-bisphosphatase

Chloroplast fructose-1,6-bisphosphatase hysteresis in response to modifiers was uncovered by carrying out the enzyme assays in two consecutive steps. The activity of chloroplast fructose-1,6-bisphosphatase, assayed at low concentrations of both fructose-1,6-bisphosphatase and Mg2+, was enhanced by preincubating the enzyme with dithiothreitol, thioredoxin f, fructose 1,6-bisphosphate, and Ca2+. In the time-dependent activation process, fructose 1,6-bisphosphate and Ca2+ could be replaced by other sugar biphosphates and Mn2+, respectively. Once activated, chloroplast fructose-1,6-bisphosphatase hydrolyzed fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate in the presence of Mg2+, Mn2+, or Fe2+. The A0.5 for fructose 1,6-bisphosphate (activator) was lowered by reduced thioredoxin f and remained unchanged when Mg2+ was varied during the assay of activity. On the contrary, the S0.5 for fructose 1,6-bisphosphate (substrate) was unaffected by reduced thioredoxin f and depended on the concentration of Mg2+. Ca2+ played a dual role on the activity of chloroplast fructose-1,6-bisphosphatase; it was a component of the concerted activation and an inhibitor in the catalytic step. Provided dithiothreitol was present, the activating effectors were not required to maintain the enzyme in the active form. Considered together these results strongly suggest that the regulation of fructose-1,6-bisphosphatase in chloroplast occurs at two different levels, the activation of the enzyme and the catalysis.     

Regulation of sedoheptulose-1,7-bisphosphatase by sedoheptulose-7-phosphate and glycerate,and of fructose-1,6-bisphosphatase by glycerate in spinach chloroplasts

Using partially purified sedoheptulose-1,7-bisphosphatase from spinach (Spinacia oleracea L.) chloroplasts the effects of metabolites on the dithiothreitoland Mg²⁺-activated enzyme were investigated. A screening of most of the intermediates of the Calvin cycle and the photorespiratory pathway showed that physiological concentrations of sedoheptulose-7-phosphate and glycerate specifically inhibited the enzyme by decreasing its maximal velocity. An inhibition by ribulose-1,5-bisphosphate was also found. The inhibitory effect of sedoheptulose-7-phosphate on the enzyme is discussed in terms of allowing a control of sedoheptulose-1,7-bisphosphate hydrolysis by the demand of the product of this reaction. Subsequent studies with partially purified fructose-1,6-bisphosphatase from spinach chloroplasts showed that glycerate also inhibited this enzyme. With isolated chloroplasts, glycerate was found to inhibit CO₂ fixation by blocking the stromal fructose-1,6-bisphosphatase. It is therefore possible that the inhibition of the two phosphatases by glycerate is an important regulatory factor for adjusting the activity of the Calvin cycle to the ATP supply by the light reaction.Abbreviations DTT dithiothreitol - FBPase fructose-1,6-bisphosphatase - Fru-1,6-P₂ fructose-1,6-bisphosphate - Fru-6-P fructose-6-phosphate - 3-PGA 3-phosphoglycerate - Ru-1,5-P₂ ribulose-1,5-bisphosphate - Ru-5-P ribulose-5-phosphate - SBPase sedoheptulose-1,7-bisphosphatase - Sed-1,7-P₂ sedoheptulose-1,7-bisphosphate - Sed-7-P sedoheptulose-7-phosphate This work was supported by the Deutsche Forschungsgemein-schaft.     

Control of Photosynthetic Sucrose Synthesis by Fructose 2,6-Bisphosphate : V. Modulation of the Spinach Leaf Cytosolic Fructose 1,6-Bisphosphatase Activity in Vitro by Substrate, Products, pH, Magnesium, Fructose 2,6-Bisphosphate, Adenosine Monophosphate, and Dihydroxyacetone Phosphate

How fructose 2,6-bisphosphate and metabolic intermediates interact to regulate the activity of the cytosolic fructose 1,6-bisphosphatase in vitro has been investigated. Mg²⁺ is required as an activator. There is a wide pH optimum, especially at high Mg²⁺. The substrate dependence is not markedly pH dependent. High concentrations of Mg²⁺ and fructose 1,6-bisphosphate are inhibitory, especially at higher pH. Fructose 2,6-bisphosphate inhibits over a wide range of pH values. It acts by lowering the maximal activity and lowering the affinity for fructose 1,6-bisphosphate, for which sigmoidal saturation kinetics are induced, but the Mg²⁺ dependence is not markedly altered. On its own, adenosine monophosphate inhibits competitively to Mg²⁺ and noncompetitively to fructose 1,6-bisphosphate. In the presence of fructose 2,6-bisphosphate, adenosine monophosphate inhibits in a fructose 1,6-bisphosphate-dependent manner. In the presence of adenosine monophosphate, fructose 2,6-bisphosphate inhibits in Mg²⁺-dependent manner. Fructose 6-phosphate and phosphate both inhibit competitively to fructose 1,6-bisphosphate. Fructose 2,6-bisphosphate does not affect the inhibition by phosphate, but weakens inhibition by fructose 6-phosphate. Dihydroxyacetone phosphate and hydroxypyruvate inhibit noncompetitively to fructose 1,6-bisphosphate and to Mg²⁺, but both act as activators in the presence of fructose 2,6-bisphosphate by decreasing the S_0.5 for fructose 1,6-bisphosphate. A model is proposed to account for the interaction between these effectors.     

Ribose 1,5-bisphosphate inhibits fructose-1,6-bisphosphatase in rat kidney cortex

Fructose-1,6-bisphosphatase is one of the regulatory enzymes of gluconeogenesis in kidney cortex. The effect of ribose 1,5-bisphosphate on fructose-1,6-bisphosphatase purified from rat kidney cortex was studied. Rat kidney cortex, fructose-1,6-bisphosphatase exhibited hyperbolic kinetics with regard to its substrate, but the activity was inhibited by ribose 1,5-bisphosphate at nanomolar concentrations. The inhibitory effect of ribose 1,5-bisphosphate on the fructose-1,6-bisphosphatase was enhanced in the presence of AMP, one of the inhibitors of fructose-1,6-bisphosphatase. Fructose-2,6-bisphosphate, which is an inhibitor of fructose-1,6-bisphosphatase, inhibited rat kidney cortex fructose-1,6-bisphosphatase activities at a low concentration of fructose-1,6-bisphosphate but a high concentration of fructose-1,6-bisphosphate relieved fructose-1,6-bisphosphatase from fructose-2,6-bisphosphate-dependent inhibition. On the contrary, fructose-1,6-bisphosphate was not effective for the recovery of fructose-1,6-bisphosphatase from ribose 1,5-bisphosphate-dependent inhibition. These results suggest that ribose 1,5-bisphosphate is a potent inhibitor and is involved in the regulation of fructose-1,6-bisphosphatase in rat kidney cortex.     

The binding of products, metal ion, and a substrate analog to rabbit liver fructose bisphosphatase.

Binding of fructose-6-P and P_i to rabbit liver fructose bisphosphatase has been analyzed in terms of four negatively cooperative binding sites per enzyme tetramer. The association of fructose-6-P occurs in the absence of divalent metal ion, although the extent of binding is increased in the order Mg²⁺ < Zn²⁺ < Mn²⁺. The binding of P_i shows an absolute requirement for divalent metal ion with Mn²⁺ being more effective than Mg²⁺. The interaction of the enzyme with the substrate analog, (α + β) methyl-d-fructofuranoside-1,6-P₂ in the presence of Mn²⁺ closely resembles that found for fructose-1,6-P₂ in the absence of Mn²⁺, although the measured constants are on average an order of magnitude smaller. Combination experiments with the three ligands show that the binding follows an identical ordered sequence, i.e., the tighter sites are initially occupied regardless of the ligand's identity. The binding of P_i or fructose-6-P is not altered by the presence of the other. Comparison of binding constant with K_i values obtained from steady-state assays permits identification of the catalytic sites expressed in the latter. The association of Mn²⁺ at the catalytic site can be induced by fructose-6-P or the substrate analog suggesting that a 1-phosphoryl group enhances but is not necessary for Mn²⁺ binding at this site. The binding of AMP is decreased in the presence of substrate analog relative to fructose-1,6-P₂, suggesting that the 2-hydroxyl serves as a “molecular signal.” From the single and combined binding experiments, a calculation of the equilibrium constant for the overall hydrolysis reaction on the enzyme surface in the presence of Mn²⁺ has been carried out and an estimate made for the Mg²⁺ case.     

Inhibition of fructose-1,6-bisphosphatase by a new class of allosteric effectors

A highly constrained pseudo-tetrapeptide (OC252-324) further defines a new allosteric binding site located near the center of fructose-1,6-bisphosphatase. In a crystal structure, pairs of inhibitory molecules bind to opposite faces of the enzyme tetramer. Each ligand molecule is in contact with three of four subunits of the tetramer, hydrogen bonding with the side chain of Asp187 and the backbone carbonyl of residue 71, and electrostatically interacting with the backbone carbonyl of residue 51. The ligated complex adopts a quaternary structure between the canonical R- and T-states of fructose-1,6-bisphosphatase, and yet a dynamic loop essential for catalysis (residues 52-72) is in a conformation identical to that of the T-state enzyme. Inhibition by the pseudo-tetrapeptide is cooperative (Hill coefficient of 2), synergistic with both AMP and fructose 2,6-bisphosphate, noncompetitive with respect to Mg2+, and uncompetitive with respect to fructose 1,6-bisphosphate. The ligand dramatically lowers the concentration at which substrate inhibition dominates the kinetics of fructose-1,6-bisphosphatase. Elevated substrate concentrations employed in kinetic screens may have facilitated the discovery of this uncompetitive inhibitor. Moreover, the inhibitor could mimic an unknown natural effector of fructose-1,6-bisphosphatase, as it interacts strongly with a conserved residue of undetermined functional significance.     

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.     

Fructose-1,6-bisphosphatase from rat liver. A comparison of the kinetics of the unphosphorylated enzyme and the enzyme phosphorylated by cyclic AMP-dependent protein kinase

A purification procedure for rat hepatic fructose-1,6-bisphosphatase, described earlier, has been improved, resulting in an enzyme preparation with a neutral pH optimum and with both phosphorylatable serine residues present. The subunit Mr was 40,000. Phosphorylation in vitro with cyclic AMP-dependent protein kinase resulted in the incorporation of 1.4 mol of phosphate/mol of subunit and led to an almost 2-fold decrease in apparent Km for fructose-1,6-bisphosphate. In contrast to yeast fructose-1,6-bisphosphatase, fructose-2,6-bisphosphate had no effect on the rate of phosphorylation or dephosphorylation of the intact enzyme. The effects of the composition of the assay medium, with regard to buffering substance and Mg2+ concentration, on the apparent Km values of phosphorylated and unphosphorylated enzyme were investigated. The kinetics of phosphorylated and unphosphorylated fructose-1,6-bisphosphatase were studied with special reference to the inhibitory effects of adenine nucleotides and fructose-2,6-bisphosphate. Unphosphorylated fructose-1,6-bisphosphatase was more susceptible to inhibition by both AMP and fructose 2,6-bisphosphate than phosphorylated enzyme, at high and low substrate concentrations. Both ATP and ADP had a similar effect on the two enzyme forms, ADP being the more potent inhibitor. Finally, the combined effect of several inhibitors at physiological concentrations was studied. Under conditions resembling the gluconeogenic state, phosphorylated fructose-1,6-bisphosphatase was found to have twice the activity of the unphosphorylated enzyme.     

Fructose-1,6-bisphosphatase in mantle of the sea mussel Mytilus galloprovincialis Lmk—I. Purification and ion requirements

• 1.1. The enzyme fructose-1,6-bisphosphatase was purified from the mantle of the sea mussel Mytilus galloprovincialis Lmk. The purified enzyme showed a single band in SDS-polyacrylamide gel electrophoresis. The mol. wt and subunit mol. wt of the enzyme were 105,000 and 27,000, respectively.
• 2.2. Divalent cations are essential for the enzyme activity. In the absence of chelating agents, FBPase 1 exhibits hyperbolic kinetics with respect to Mn²⁺, Zn²⁺ and Mg²⁺. The K_m for Mg²⁺ is lower than the physiological concentration of cation in the tissue, whereas its K_m for Mn²⁺ and Zn²⁺ is greater than the respective in vivo concentrations.
• 3.3. The joint action of Mg²⁺ and Zn²⁺ increases the affinity of the enzyme for the substrate Fru-1,6-P₂, though V_max is reduced.
• 4.4. Na⁺ strongly inhibits the enzyme even at very low concentrations. K⁺ has no effect whatsoever.

     

Isolation and characterization of an allelic cDNA for human muscle fructose-1,6-bisphosphatase

By applying a newly developed method, cDNAs for the human muscle isoform of fructose-1,6-bisphosphatase were isolated from phage- and plasmid-derived libraries. From these cDNAs and an EST clone, a composite sequence (1302 bp) was deduced that contains an open reading frame encoding a polypeptide of 339 amino acids with an estimated molecular weight of 36 755. After overexpression in E. coli, recombinant human muscle fructose-1,6-bisphosphatase was found to be active in cell-free extracts and could be strongly inhibited by AMP and fructose 2,6-bisphosphate. Sequence comparisons revealed that (1) all amino acids thought to be in contact with substrate molecules, regulatory molecules or metal ions in mammalian liver fructose-1,6-bisphosphatases are, with one exception, conserved in the human muscle enzyme and (2) the human muscle isoform is more homologous to the mouse intestine fructose-1,6-bisphosphatase than to the mammalian liver isoform. This is the first report of the cloning and expression of a muscle fructose-1,6-bisphosphatase isoenzyme.     

Variation of (2''-5'')oligo(adenylate) synthetase activity during rat-liver regeneration

The inhibitory effect of fructose 2,6-biphosphate on fructose 1,6-bisphosphatase was reinvestigated in order to solve the apparent contradiction between competition with the substrate and the synergism with AMP, a strictly noncompetitive inhibitor. The effect of fructose 2,6-bisphosphate was compared to that of other ligands of the enzyme, which, like the substrate and methyl (alpha + beta)fructofuranoside 1,6-bisphosphate bind to the active site or which, like AMP, bind to an allosteric site. An increase in temperature or pH, or the presence of sulfosalicylate, lithium or higher concentrations of magnesium as well as partial proteolysis by subtilisin increased [I]0.5 for fructose 2,6-bisphosphate and AMP without affecting Km. With the exception of the pH change, all these conditions were also without effect on the affinity of the enzyme for the competitive inhibitor, methyl (alpha + beta)fructofuranoside 1,6-bisphosphate. These observations can be explained by assuming that fructose 2,6-bisphosphate has no affinity for the active site of fructose 1,6-bisphosphatase but binds to an allosteric site which is different from the AMP site. Fructose 2,6-bisphosphate is therefore classified as an allosteric competitive inhibitor and a model is proposed which explains its synergism with AMP as well as the various cooperative effects.     

Binding of Zn2+ to rabbit muscle fructose 1,6-bisphosphatase and its effect on the catalytic properties.

At low concentrations (<1 μM), and in the presence of Mg²⁺, Zn²⁺ inhibits the activity of rabbit muscle fructose 1,6-bisphosphatase (EC 3.1.3.11). At higher concentrations Zn²⁺ can replace Mg²⁺ as the activating cation. The inhibitory effects of Zn²⁺ are associated with its binding to 4 high-affinity sites (1 per subunit). Binding to a second set of 4 sites requires the presence of the substrate, fructose 1,6-bisphosphate, and binding of Zn²⁺ to this set of sites restores the catalytic activity. In the absence of EDTA, Zn²⁺ is a better activating cation than Mg²⁺. The muscle enzyme differs from rabbit liver fructose 1,6-bisphosphatase in the number of binding sites (8 as compared to 12 for the rabbit liver enzyme) and in showing higher activity with Zn²⁺ as the activating cation. The results suggest that Zn²⁺ may be the physiological activator.