Fructose-1,6-biphosphatase of the small intestine. Purification and comparison with liver and muscle fructose-1,6-bisphosphatases.

The occurrence of specific fructose-1,6-bisphosphatase [D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11] (Fru-1,6-P2ase) in the small intestine was confirmed. 1. Fru-1,6-P2ase was isolated from mouse small intestine by a simple method. The isolated enzyme preparation was an electrophoretically homogeneous protein. 2. The molecular weight and subunit molecular weight were 140,000 and 38,000, respectively. 3. The intestinal enzyme was electrophoretically distinct from the liver enzyme. 4. The kinetic properties of the purified intestinal enzyme were compared with those of the mouse liver and muscle enzymes. 5. Mouse intestinal and muscle Fru-1,6-P2ases hydrolyzed ribulose-1,5-bisphosphate in addition to fructose-1,6-bisphosphate and sedoheptulose-1,7-bisphosphate.     

Different sensitivities of mutants and chimeric forms of human muscle and liver fructose-1,6-bisphosphatases towards AMP

AMP is an allosteric inhibitor of human muscle and liver fructose-1,6-bisphosphatase (FBPase). Despite strong similarity of the nucleotide binding domains, the muscle enzyme is inhibited by AMP approximately 35 times stronger than liver FBPase: I0.5 for muscle and for liver FBPase are 0.14 microM and 4.8 microM, respectively. Chimeric human muscle (L50M288) and chimeric human liver enzymes (M50L288), in which the N-terminal residues (1-50) were derived from the human liver and human muscle FBPases, respectively, were inhibited by AMP 2-3 times stronger than the wild-type liver enzyme. An amino acid exchange within the N-terminal region of the muscle enzyme towards liver FBPase (Lys20-->Glu) resulted in 13-fold increased I0.5 values compared to the wild-type muscle enzyme. However, the opposite exchanges in the liver enzyme (Glu20-->Lys and double mutation Glu19-->Asp/Glu20-->Lys) did not change the sensitivity for AMP inhibition of the liver mutant (I0.5 value of 4.9 microM). The decrease of sensitivity for AMP of the muscle mutant Lys20-->Glu, as well as the lack of changes in the inhibition by AMP of liver mutants Glu20-->Lys and Glu19-->Asp/Glu20-->Lys, suggest a different mechanism of AMP binding to the muscle and liver enzyme.     

Amino acid sequence homology among fructose-1,6-bisphosphatases

The hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate is a key reaction of carbohydrate metabolism. The enzyme that catalyzes this reaction, fructose-1,6-bisphosphatase, appears to be present in all forms of living organisms. Regulation of the enzyme activity, however, occurs by a variety of distinct mechanisms. These include AMP inhibition (most sources), cyclic AMP-dependent phosphorylation (yeast), and light-dependent activation (chloroplast). In the present studies, we have made a comparison of the primary structure of mammalian fructose-1,6-bisphosphatase with the sequence of peptides isolated from the yeast Saccharomyces cerevisiae, Escherichia coli, and spinach chloroplast enzymes. Our results demonstrate a high degree of sequence homology, suggesting a common evolutionary origin for all fructose-1,6-bisphosphatases.     

Effect of Mn2+ on fructose 2,6-bisphosphate inhibition of mouse liver, intestinal, and muscle fructose-1,6-bisphosphatases

Fructose 2,6-bisphosphate inhibited all three fructose-1,6-bisphosphatases from the liver, intestine, and muscle of the mouse. The sensitivity of the liver enzyme to the inhibitor was significantly diminished when Mg2+ was replaced by Mn2+ as the activating cation. Inhibition of the liver enzyme by fructose 2,6-bisphosphate decreased as the concentration of the metal activator, Mn2+ or Mg2+, increased. The respective I50 values obtained by extrapolation of metal ion concentrations to zero were 40 microM with Mn2+ and 0.25 microM with Mg2+. The extent of desensitization to either fructose 2,6-bisphosphate or AMP inhibition by Mn2+ decreased in the order of the liver, intestine, and muscle enzyme. Only in the case of the liver enzyme was the substrate cooperativity induced by fructose 2,6-bisphosphate in the presence of Mg2+. In all three isoenzymes from the mouse, fructose 2,6-bisphosphate greatly potentiated the AMP inhibition of the enzyme in the presence of either Mg2+ or Mn2+. The liver enzyme with Mn2+ in addition to Mg2+ was still active in the presence of less than 1 microM fructose 2,6-bisphosphate, even though AMP was present at 100-200 microM.     

Plant fructose-1,6-bisphosphatases: characteristics and properties.

In this minireview the properties and characteristics of plant fructose-1,6-bisphosphatases (D-fructose-1,6-bisphosphatase 1-phosphohydrolase, EC 3.1.3.11) are discussed. The properties and characteristics of the chloroplastic and cytoplasmic forms of the enzyme are reviewed. For purposes of comparison some reference is made to fructose-1,6-bisphosphatases from other species.     

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.     

Antigenic relationships between chloroplast and cytosolic fructose-1,6-bisphosphatases.

Cytosolic fructose-1,6-biphosphatases (FBPase, EC 3.1.3.11) from pea (Pisum sativum L. cv Lincoln) and spinach (Spinacia oleracea L. cv Winter Giant) did not cross-react by double immunodiffusion and western blotting with either of the antisera raised against the chloroplast enzyme of both species; similarly, pea and spinach chloroplast FBPases did not react with the spinach cytosolic FBPase antiserum. On the other hand, spinach and pea chloroplast FBPases showed strong cross-reactions against the antisera to chloroplast FBPases, in the same way that the pea and spinach cytosolic enzymes displayed good cross-reactions against the antiserum to spinach cytosolic FBPase. Crude extracts from spinach and pea leaves, as well as the corresponding purified chloroplast enzymes, showed by western blotting only one band (44 and 43 kD, respectively) in reaction with either of the antisera against the chloroplast enzymes. A unique fraction of molecular mass 38 kD appeared when either of the crude extracts or the purified spinach cytosolic FBPase were analyzed against the spinach cytosolic FBPase antiserum. These molecular sizes are in accordance with those reported for the subunits of the photosynthetic and gluconeogenic FBPases. Chloroplast and cytosolic FBPases underwent increasing inactivation when increasing concentrations of chloroplast or cytosolic anti-FBPase immunoglobulin G (IgG), respectively, were added to the reaction mixture. However, inactivations were not observed when the photosynthetic enzyme was incubated with the IgG to cytosolic FBPase, or vice versa. Quantitative results obtained by enzyme-linked immunosorbent assays (ELISA) showed 77% common antigenic determinants between the two chloroplast enzymes when tested against the spinach photosynthetic FBPase antiserum, which shifted to 64% when assayed against the pea antiserum. In contrast, common antigenic determinats between the spinach cytosolic FBPase and the two chloroplast enzymes were less than 10% when the ELISA test was carried out with either of the photosynthetic FBPase antisera, and only 5% when the assay was performed with the antiserum to the spinach cytosolic FBPase. These results were supported by sequencing data: the deduced amino acid sequence of a chloroplast FBPase clone isolated from a pea cDNA library indicated a 39,253 molecular weight protein, with a homology of 85% with the spinach chloroplast FBPase but only 48.5% with the cytosolic enzyme from spinach.     

Inhibition of fructose-1,6-bisphosphate aldolase from rabbit muscle and Bacillus stearothermophilus

Phosphoglycollohydroxamic acid and phosphoglycollamide are inhibitors of rabbit muscle fructose-1,6-bisphosphate aldolase. The binding dissociation constants determined by enzyme inhibition and protein fluorescence quenching suggest that two distinct enzyme inhibitor complexes may be formed. The binding dissociation constants of the two inhibitors to Bacillus stearothermophilus cobalt (II) fructose-1,6-bisphosphate aldolase have also been determined. The hydroxamic acid is an exceptionally potent inhibitor (Ki = 1.2 nM) probably due to direct chelation with Co(II) at the active site. The inhibition, however, is time-dependant and the association and dissociation constants have been estimated. Ethyl phosphoglycollate irreversibly inhibits rabbit muscle fructose-1,6-bisphosphate aldolase in the presence of sodium borohydride, presumably by forming a stable secondary amine through the active-site lysine reside. A new condensation assay for fructose-1,6-bisphosphate aldolases has been developed which is more sensitive than currently used assay procedures.     

Regulation of rabbit liver fructose-1,6-bisphosphatase by metals, nucleotides, and fructose 2,6-bisphosphate as determined from fluorescence studies

The fluorescent nucleotide analogue formycin 5'-monophosphate (FMP) inhibits rabbit liver fructose-1,6-bisphosphatase (I50 = 17 microM, Hill coefficient = 1.2), as does the natural regulator AMP (I50 = 13 microM, Hill coefficient = 2.3), but exhibits little or no cooperativity of inhibition. Binding of FMP to fructose-1,6-bisphosphatase can be monitored by the increased fluorescence emission intensity (a 2.7-fold enhancement) or the increased fluorescence polarization of the probe. A single dissociation constant for FMP binding of 6.6 microM (4 sites per tetramer) was determined by monitoring fluorescence intensity. AMP displaces FMP from the enzyme as evidenced by a decrease in FMP fluorescence and polarization. The substrates, fructose 6-phosphate and fructose 1,6-bisphosphate, and inhibitors, methyl alpha-D-fructofuranoside 1,6-bisphosphate and fructose 2,6-bisphosphate, all increase the maximal fluorescence of enzyme-bound FMP but have little or no effect on FMP binding. Weak metal binding sites on rabbit liver fructose-1,6-bisphosphatase have been detected by the effect of Zn2+, Mn2+, and Mg2+ in displacing FMP from the enzyme. This is observed as a decrease in FMP fluorescence intensity and polarization in the presence of enzyme as a function of divalent cation concentration. The order of binding by divalent cations is Zn2+ = Mn2+ greater than Mg2+, and the Kd for Mn2+ displacement of FMP is 91 microM. Methyl alpha-D-fructofuranoside 1,6-bisphosphate, as well as fructose 6-phosphate and inorganic phosphate, enhances metal-mediated FMP displacement from rabbit liver fructose-1,6-bisphosphatase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural changes of rabbit liver fructose-1,6-bisphosphatase: the effect of urea and subtilisin digestion

At pH 6.3 both the native and subtilisin-digested fructose-1,6-bisphosphatase (Fru-P2-ase) molecules exhibit four fast-reacting thiol groups. The kinetic analysis shows that the pK value for the reaction of these thiols is 8.1. The increase of pH from 6.3 to 9.3 results in an uncovering of the remaining 20 thiol groups. In subtilisin-cleaved enzyme the rate of reaction of SH groups is considerably higher than in the native enzyme at pH 9.3, indicating changes in the microenvironments around thiols upon modification. A fluorescent label inserted on a fast-reacting SH group and neighboring NH2 group shifts the pH optimum of the enzyme to alkaline region and decreases its sensitivity toward AMP. Spectral analysis of labeled enzyme indicates that the labeled region of protein is more hydrophilic upon proteolytic digestion. It is concluded that a molecule of subtilisin-digested enzyme has a more relaxed structure than the native enzyme. The relaxation of the enzyme to a new conformation is reflected by urea addition, which mimics the effect of subtilisin digestion. Correlation of enzyme activity versus its sensitivity toward AMP (I 0.5), shows that at low concentrations of urea the active-site region at pH 6.3 is more affected than the region of AMP binding.     

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.     

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.     

Complex type of kinetics of fructose-1,6-diphosphate from the rat and rabbit liver

Studies on rat and rabbit liver fructose 1.6-bisphosphatase inhibition by AMP showed that with an increase in EDTA concentration the hyperbolic AMP inhibition curve is transformed into a sigmoidal one. At intermediate EDTA concentrations, the kinetic curves have a plateau. The appearance of the intermediate plateau may be due to the superposition of kinetic curves corresponding to two enzyme forms simultaneously present in the assay mixture. One of these forms deprived of endogenous Me2+ (presumably Zn2+) is inhibited by AMP in a cooperative manner, while the other one retains Me2+ which prevents the cooperative response of the enzyme to AMP.     

Preliminary X-ray crystallographic studies of pig kidney fructose-1,6-bisphosphatase

Preliminary x-ray data have been obtained from large single crystals of pig kidney fructose-1,6-bisphosphatase, grown from polyethylene glycol. The crystals have the symmetry of space group P3(1)21 or its enantiomorph P3(2)21, contain two subunits of the 146,000-dalton tetramer/asymmetric unit, and diffract to 2.9-A resolution on still photographs. The unit cell dimensions are a = b = 132.5 A and c = 68.0 A. Small single crystals have been grown in the presence of the inhibitor fructose 2,6-bisphosphate, with and without the allosteric effector AMP added. Crystals grown in the presence of both ligands are isomorphous with native crystals and generate diffraction patterns that show significant intensity changes.     

Concentration of liver and kidney fructose-1,6-diphosphatase determined by specific radioimmunoassay

A radioimmunoassay for liver fructose-1,6-diphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrlase, EC 3.1.3.11) has been developed based on maintenance of its tetrameric structure and immunologic integrity after iodination by the Bolton-Hunter technique. The assay detected as little as 2 ng of standard enzyme. Nonspecific interference by tissue components did not occur. Enzyme concentration (mumol/1000 g tissue wet weight) was measured in tissue extracts of 49 rabbits subjected to a variety of conditions. In animals fed a 'balanced' diet containing 50--60% carbohydrate (by weight), the concentration in liver was 3.4 microM +/- 0.3. After fasts of 48, 72, or 96 h, the concentration in liver increased approximately 1.4-fold. A high-fat diet did not alter the concentration significantly but a high-protein diet caused an increase of 2.1-fold to 7.2 microM +/- 1.4. The greatest concentrations, 8.7 microM +/- 1.9, were observed in the livers of severely diabetic rabbits. The increase paralleled the increasing severity of diabetes and provides one explanation for the augmented gluconeogenesis which occurs in the diabetic state. Changes were less marked in kidney. The greatest apparent incrase, from 2.6 microM +/- 1.1 in the normal fed rabbit to 4.7 microM +/- 2.8, occurred in the severely diabetic animal. However, variation was sufficiently great in kidney to render apparent increases during fasting, protein feefing and diabetes statistically insignificant. For the most part changes in assayable activity followed changes in enzyme concentration except in the rabbits maintained on high-protein diets. In these, liver enzyme concentration increased by 2.4-fold whereas activity increased by only 1.3-fold, and the kidney enzyme concentration increased 1.3-fold whereas activity decreased by 20%.     

Size-dependent allosteric effects of monovalent cations on rabbit liver fructose-1,6-bisphosphatase.

Effects of monovalent cations on the neutral rabbit liver fructose-1,6-bisphosphatase are multifunctional and dependent on their nonhydrated ionic size. (a) The maximal velocity is increased by addition of monovalent cations with the optimum stimulation occurring with a nonhydrated ionic radius of 1.2 A in the presence of a chelating agent such as EDTA. (B) Activation curves are sigmoidal with n values varying from 1.5 to 2.3 as ionic radius of monovalent cation increases. The apparent Ka values from 16.0 to 180 mM, obtained for various monovalent cations, have a linear relationship to ionic radii of cations. (c) At lower concentrations of fructose 1,6-bisphosphate monovalent cations show the inhibitory effect and the apparent Km for fructose 1,6-bisphosphate is increased as the concentration of monovalent cation is increased. A linear relationship is obtained between the slopes of increase in the Km and the reciprocals of ionic volume of monovalent cations. (d) The apparent Ka for Mg2+ is also increased as the concentration of monovalent cation is increased, and a linear relationship is obtained again between the increases in Ka and the reciprocals of ionic volume of monovalent cations. The cooperative nature for Mg2+ saturation is decreased as the Ka increases. (e) The apparent Ki for AMP is also linearly altered as the concentration of monovalent cation is varied. However, the alteration of the Ki is unusual, that is, the smaller cations than K+ increase the Ki (Li+ greater than Na+ greater than NH4+), whereas the larger cations decrease the value ((CH2CH2OH)3N+ greater than Cs+ greater than Rb+). The effect of K+ is insignificant. Alterations in the Ki are also linearly related to the reciprocals of ionic volume of monovalent cations. The cooperative nature for AMP inhibition is decreased or increased as the Ki increased or decreased. (f) In the absence of the chelating agent, the curves for Mg2+ saturation and AMP inhibition were hyperbolic without monovalent cations. By addition of monovalent cation the Ka for Mg+2+ or Ki for AMP is increased and cooperative natures for binding of both ligands are induced. For nonspherical monovalent cations, the application of "functional ionic radius" is proposed. Functional ionic radii of NH4+, (CH2OH)3CNH3+, and (CH2CH2OH)3N+ are estimated to be 1.17, 2.55, and 2.87 A, respectively. The presence of two distinct sites for the actions of monovalent cations is suggested.     

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.     

Construction of chimeric cytosolic fructose-1,6-bisphosphatases by insertion of a chloroplastic redox regulatory cluster

In order to transform cytosolic fructose-1,6-bisphosphatases (FBPase)(EC 3.1.3.11) into potential reductively-modulated chloroplast-type enzymes, we have constructed four chimeric FBPases, which display structural viability as deduced by previous modelling. In the X1-type BV1 and HL1 chimera the N-half of cytosolic sugar beet (Beta vulgaris L.) and human FBPases was fused with the C-half of the pea (Pisum sativum L.) chloroplast enzyme, which carries the cysteine-rich light regulatory sequence. In the X2-type BV2 and HL2 chimera this regulatory fragment was inserted in the corresponding site of the sugar beet cytosolic and human enzymes. Like the plant cytosolic FBPases, the chimeric enzymes show a low rise of activity by dithiothreitol. Both BV1 and BV2, but not HL1 and HL2, display a negligible activation by Trx f, but neither of them by Trx m. Antibodies raised against the pea chloroplast enzyme showed a positive reaction against the four chimeric FBPases and the human enzyme, but not against the sugar beet one. The four chimera display typical kinetics of cytosolic FBPases, with Km values in the 40-140 microM range. We conclude the existence of a structural capacity of cytosolic FBPases for incorporating the redox regulatory cluster of the chloroplast enzyme. However, the ability of these chimeric FBPases for an in vitro redox regulation seems to be scarce, limiting their use from a biotechnology standpoint in in vivo regulation of sugar metabolism.