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.     

Kinetics of the conformational transition of the spinach chloroplast fructose-1,6-bisphosphatase induced by fructose 2,6-bisphosphate.

The activation of oxidized chloroplast fructose-1,6-bisphosphatase by fructose 2,6-bisphosphate and magnesium previously described at pH 7.5 [Soulié et al. (1988) Eur. J. Biochem. 176, 111-117] has now been studied at pH 8, the pH which prevails under light conditions in the chloroplast stroma. The process obeys a hysteretic mechanism but the rate of activation is considerably increased with half-times down to 50 s and the apparent dissociation constant of fructose 2,6-bisphosphate from the enzyme is lowered from 1 mM at pH 7.5 to 3.3 microM at pH 8. The process is strictly metal-dependent with a half-saturation concentration of 2.54 mM for magnesium. The conformational transition postulated in our hysteretic model has been investigated through both the spectrophometric and chemical modification approaches. The activation of the enzyme by fructose 2,6-bisphosphate in the presence of magnesium results in a slow modification of the ultraviolet absorption spectrum of the enzyme with an overall increase of 3% at 290 nm. The same treatment leads to the protection of two free sulfhydryls and an increased reactivity of one sulfhydryl group/enzyme monomer to modification by 5,5'-dithiobis(2-nitrobenzoic acid). The titration of the exposed cysteinyl residue prevents the relaxation of enzyme species induced by fructose 2,6-bisphosphate to the native form. The activation of chloroplast fructose-1,6-bisphosphatase by fructose 2,6-bisphosphate is discussed both with respect to the understanding of the overall regulation properties of the enzyme and to a possible physiological significance of this process.     

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.     

Allosteric inhibition of Dictyostelium discoideum fructose-1,6-bisphosphatase by fructose 2,6-bisphosphate

It has been found that the inhibition of Dictyostelium discoideum fructose-1,6-bisphosphatase by fructose 2,6-P2 greatly diminished when the pH was raised to the range 8.5-9.5, which resulted in a marked decrease of the affinity for the inhibitor with no change in the Km for the substrate. This provides evidence for the involvement of an allosteric site for fructose 2,6-P2. Moreover, the fact that excess substrate inhibition also decreased at the pH values for minimal fructose 2,6-P2 inhibition, and was essentially abolished in the presence of fructose 2,6-P2, strongly suggests that this inhibition takes place by binding of fructose 1,6-P2 as a weak analogue of the physiological effector fructose 2,6-P2.     

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.     

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.     

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.     

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.     

Lysine 274 is essential for fructose 2,6-bisphosphate inhibition of fructose-1,6-bisphosphatase.

Lysine 274 is conserved in all known fructose-1,6-bisphosphatase sequences. It has been implicated in substrate binding and/or catalysis on the basis of reactivity with pyridoxal phosphate as well as by x-ray crystallographic analysis. Lys274 of rat liver fructose-1,6-bisphosphatase was mutated to alanine by the polymerase chain reaction, and the T7-RNA polymerase-transcribed construct containing the mutant sequence was expressed in Escherichia coli. The mutant and wild-type forms of the enzyme were purified to homogeneity, and their specific activity, substrate dependence, and inhibition by fructose 2,6-bisphosphate and AMP were compared. While the mutant exhibited no change in maximal velocity, its Km for fructose 1,6-bisphosphate was 20-fold higher than that of the wild-type, and its Ki for fructose 2,6-bisphosphate was increased 1000-fold. Consistent with the unaltered maximal velocity, there were no apparent difference between the secondary structure of the wild-type and mutant enzyme forms, as measured by circular dichroism and ultraviolet difference spectroscopy. The Ki for the allosteric inhibitor AMP was only slightly increased, indicating that Lys274 is not directly involved in AMP inhibition. Fructose 2,6-bisphosphate potentiated AMP inhibition of both forms, but 500-fold higher concentrations of fructose 2,6-bisphosphate were needed to reduce the Ki for AMP for the mutant compared to the wild-type. However, potentiation of AMP inhibition of the Lys274----Ala mutant was evident at fructose 2,6-bisphosphate concentrations (approximately 100 microM) well below those that inhibited the enzyme, which suggests that fructose 2,6-bisphosphate interacts either with the AMP site directly or with other residues involved in the active site-AMP synergy. The results also demonstrate that although Lys274 is an important binding site determinant for sugar bisphosphates, it plays a more significant role in binding fructose 2,6-bisphosphate than fructose 1,6-bisphosphate, probably because it binds the 2-phospho group of the former while other residues bind the 1-phospho group of the substrate. It is concluded that the enzyme utilizes Lys274 to discriminate between its substrate and fructose 2,6-bisphosphate.     

Substrate form of D-frutose 1,6-bisphosphate utilized by fructose 1,6-bisphosphatase.

Rapid quench kinetic experiments on fructose 1,6-bisphosphatase demonstrate a stereospecificity for the alpha anomer of fructose 1,6-bisphosphate relative to the beta configuration. The beta anomer is only utilized after mutarotation to the alpha form in a process that is not enzyme catalyzed. Studies employing analogues of the acyclic keto configuration indicate that the keto form is utilized at a rate less than 5% that of the alpha anomer, a finding also confirmed by computer simulation of the rapid quench data. Chemical trapping experiments of the keto analogue, xylulose 1,5-bisphosphate, and the normal substrate suggest that interconversion of the acyclic and anomeric configurations is retarded by their binding to the enzyme. A hypothesis is advanced attributing substrate inhibition of fructose 1,6-bisphosphatase to possible binding of the keto species.     

Reciprocal regulation of fructose 1,6-bisphosphatase and phosphofructokinase by fructose 2,6-bisphosphate in swine kidney

The effect of fructose 2,6-P₂, AMP and substrates on the coordinate inhibition of FBPase and activation of PFK in swine kidney has been examined. Fructose 2,6-P₂ inhibits the activity of FBPase and stimulates the activity of PFK in the presence of inhibitory concentrations of ATP. Under similar conditions 2.2 μM fructose 2,6-P₂ was required for 50% inhibition of FBPase and 0.04 μM fructose 2,6-P₂ restored 50% of the activity of PFK. Fructose 2,6-P₂ also enhanced the allosteric activation of PFK by AMP and it increased the extent of inhibition of FBPase by AMP. Fructose 2,6-P₂, AMP and fructose 6-P act cooperatively to stimulate the activity of PFK whereas the same latter two effectors and fructose 1,6-P₂ inhibit the activity of FBPase. Taken collectively, these results suggest that an increase in the intracellular level of fructose 2,6-P₂ during gluconeogenesis could effectively overcome the inhibition of PFK by ATP and simulataneously inactivate FBPase. When the level of fructose 2,6-P₂ is low, a glycolytic state would be restored, since under these conditions PFK would be inhibited by ATP and FBPase would be active.     

Selective thiol group modification renders fructose-1,6-bisphosphatase insensitive to fructose 2,6-bisphosphate inhibition

Limited treatment of native pig kidney fructose-1,6-bisphosphatase (50 microM enzyme subunit) with [14C]N-ethylmaleimide (100 microM) at 30 degrees C, pH 7.5, in the presence of AMP (200 microM) results in the modification of 1 reactive cysteine residue/enzyme subunit. The N-ethylmaleimide-modified fructose-1,6-bisphosphatase has a functional catalytic site but is no longer inhibited by fructose 2,6-bisphosphate. The enzyme derivative also exhibits decreased affinity toward Mg2+. The presence of fructose 2,6-bisphosphate during the modification protects the enzyme against the loss of fructose 2,6-bisphosphate inhibition. Moreover, the modified enzyme is inhibited by monovalent cations, as previously reported (Reyes, A., Hubert, E., and Slebe, J.C. (1985) Biochem. Biophys. Res. Commun. 127, 373-379), and does not show inhibition by high substrate concentrations. A comparison of the kinetic properties of native and N-ethylmaleimide-modified fructose-1,6-bisphosphatase reveals differences in some properties but none is so striking as the complete loss of fructose 2,6-bisphosphate sensitivity. The results demonstrate that fructose 2,6-bisphosphate interacts with a specific allosteric site on fructose-1,6-bisphosphatase, and they also indicate that high levels of fructose 1,6-bisphosphate inhibit the enzyme by binding to this fructose 2,6-bisphosphate allosteric site.     

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.     

On the mechanism of inhibition of fructose 1,6-bisphosphatase by fructose 2,6-bisphosphate

The inhibition of rabbit liver fructose 1,6-bisphosphatase (EC 3.1.3.11) by fructose 2,6-bisphosphate (Fru-2,6-P₂) is shown to be competitive with the substrate, fructose 1,6-bisphosphate (Fru-1,6-P₂), with K_i for Fru-2,6-P₂ of approximately 0.5 μm. Binding of Fru-2,6-P₂ to the catalytic site is confirmed by the fact that it protects this site against modification by pyridoxal phosphate. Inhibition by Fru-2,6-P₂ is enhanced in the presence of a noninhibitory concentration (5 μm) of the allosteric inhibitor AMP and decreased by modification of the enzyme by limited proteolysis with subtilisin. Fru-2,6-P_2, unlike the substrate Fru-1,6-P₂, protects the enzyme against proteolysis by subtilisin or lysosomal proteinases.     

Effects of pH and fructose 2,6-bisphosphate on oxidized and reduced spinach chloroplastic fructose-1,6-bisphosphatase

This report describes the effects of pH and fructose 2,6-bisphosphate (an analog of fructose 1,6-bisphosphate) on the activity of oxidized and reduced fructose-1,6-bisphosphatase from spinach chloroplasts. Studies were carried out with either fructose 1,6-bisphosphate, the usual substrate, or sedoheptulose 1,7-bisphosphate, an alternative substrate. The reduction of the oxidized enzyme is achieved by a thiol/disulfide interchange. The pK values relative to each redox form for the same substrate (either fructose 1,6-bisphosphate or sedoheptulose 1,7-bisphosphate) are identical, suggesting the same site for both substrates on the active molecule. The finding that the analog (fructose 2,6-bisphosphate) behaves like a competitive inhibitor for both substrates also favours this hypothesis. The inhibitory effect of this sugar is more important when the enzyme is reduced than when it is oxidized. The shift in the optimum pH observed when [Mg2+] was raised is interpreted as a conformational change of oxidized enzyme demonstrated by a change in fluorescence. The reduced and oxidized forms have the same theoretical rates relative to both substrates, but the reduced form has an observed Vmax which is 60% of the theoretical Vmax while that of the oxidized form is only 37% of the theoretical Vmax. The reduced enzyme appears more efficient than the oxidized one in catalysis.     

Biphasic effect of fructose 2,6-bisphosphate on the liver fructose-1,6-bisphosphatase: mechanistic and physiological implications

Fructose 2,6-bisphosphate has been claimed to be both a substrate analogue and an allosteric inhibitor of fructose-1,6-bisphosphatase. The results reported here show that fructose 2,6-bisphosphate can be both an inhibitor and an activator of the enzyme, depending on the substrate concentration. This biphasic behaviour at saturating concentrations of substrate can only be due to an allosteric effect. In addition to the mechanistic implication it is possible that this finding may have physiological meaning.     

The site of substrate and fructose 2,6-bisphosphate binding to rabbit liver fructose-1,6-bisphosphatase

The binding site(s) in rabbit liver fructose-1,6-bisphosphatase for the active site binding ligand, fructose 6-phosphate, and the inhibitor, fructose 2,6-bisphosphate, have been investigated by using nuclear magnetic resonance spectroscopy. The distance from a nitroxide spin label to the bound ligands and the distance from the structural metal site to the bound ligands are about the same within experimental error. These data indicate that the two ligands probably bind at the active site in the rabbit liver enzyme.     

Influence of fructose 2,6-bisphosphate on the phosphofructokinase/fructose 1,6-bisphosphatase cycle

In a reconstituted enzyme system multiple stationary states and oscillatory motions of the substrate cycle catalyzed by phosphofructokinase and fructose 1,6-bisphosphatase are significantly influenced by fructose 2,6-bisphosphate. Depending on the initial conditions, fructose 2,6-bisphosphate was found either to generate or to extinguish oscillatory motions between glycolytic and gluconeogenic states. In general, stable glycolytic modes are favored because of the efficient activation of phosphofructokinase by this effector. The complex effect of fructose 2,6-bisphosphate on the rate of substrate cycling correlates with its synergistic cooperation with AMP in the activation of phosphofructokinase and inhibition of fructose 1,6-bisphosphatase.