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At least 253 records · Page 14Linked to original sources
PubMed1995

Expression of bovine heart fructose 6-phosphate,2-kinase:fructose 2,6-bisphosphatase and determination of the role of the carboxyl terminus by mutagenesis.

Bovine heart fructose 6-P,2-kinase:fructose 2,6-bisphosphatase was expressed in Escherichia coli. In order to determine the role of the carboxyl-terminal peptide, 49 and 78 amino acids from the C-terminus were deleted using oligonucleotide-directed mutagenesis. The expressed wild-type and mutant enzymes were purified to homogeneity, and the steady-state kinetics of the mutant enzymes were compared to those of the wild-type enzyme. Deletion of 49 residues (Del 49) resulted in a 35% decrease in KmFru6P, a 36% increase in Vmax, and a 2-fold increase in Kcat/Km of the kinase. There was no change in the kinetic properties of the phosphatase activity. Deletion of 78 residues (Del 78) resulted in a 4.5-fold decrease in KmFru6P, a 2.5-fold increase in Vmax, a 12-fold increase in kcat/Km of the kinase, and a 3-fold increase in kcat/Km of the phosphatase. Phosphorylation of the wild-type and Del 49 enzymes resulted in decreased KmFru6P and activation of the kinase without affecting the phosphatase activity. Thermal inactivation rates of the wild-type and Del 49 enzymes were similar, but the rate of Del 78 was more rapid. The phosphorylated wild-type and Del 49 enzymes were more sensitive to thermal inactivation than the dephospho forms. Urea inactivation of the kinase and phosphatase of wild-type and Del 49 were similar, but Del 78 was more sensitive to urea. All phosphorylated enzymes were more susceptible to urea inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1980

Aldehyde-induced adenosine triphosphatase activities of fructose 6-phosphate and fructose kinases.

Chitose-6-P (2,5-anhydromannose-6-P) induces ATPase activity of fructose-6-P kinase with a Vmax 2-3% that of the normal kinase reaction with fructose-6-P or 2,5-anhydromannitol. Chitose (and presumably also chitose-6-P) is 52% hydrated in water while chitose deuterated at C-1 is 60% hydrated because of the equilibrium isotope effect of 0.73 on aldehyde hydration. Deuterated chitose-6-P gave a normal isotope effect on V/K of 1.23, but no effect on Vmax, showing that the free aldehyde is the activator and the hydrated form does not bind appreciably. With fructokinase, chitose can act either as a substrate, being phosphorylated at C-6 when adsorbed with C-6 next to MgATP, or as an inducer of ATPase activity when adsorbed with C-1 next to MgATP. The ATPase has a rate about 25% that of the kinase.

Adenosine Triphosphatases↗
PubMed1997

The active sites of fructose 6-phosphate,2-kinase: fructose-2, 6-bisphosphatase from rat testis. Roles of Asp-128, Thr-52, Thr-130, Asn-73, and Tyr-197.

To investigate the role in catalysis and/or substrate binding of the Walker motif residues of rat testis fructose 6-phosphate, 2-kinase:fructose-2,6-bisphosphatase (Fru 6-P,2-kinase:Fru-2,6-Pase), we have constructed and characterized mutant enzymes of Asp-128, Thr-52, Asn-73, Thr-130, and Tyr-197. Replacement of Asp-128 by Ala, Asn, and Ser resulted in a small decrease in Vmax and a significant increase in Km values for both substrates. These mutants exhibited similar pH activity profiles as that of the wild type enzyme. Mutation of Thr-52 to Ala resulted in an enzyme with an infinitely high Km for both substrates and an 800-fold decreased Vmax. Substitution of Asn-73 with Ala or Asp caused a 100- and 600-fold increase, respectively in KFru 6-P with only a small increase in KATP and small changes in Vmax. Mutation of Thr-130 caused small changes in the kinetic properties. Replacement of Tyr-197 with Ser resulted in an enzyme with severely decreased binding of Fru 6-P with 3-fold decreased Vmax. A fluorescent analog of ATP, 2'(3')-O-(N-methylanthraniloyl)ATP (mant-ATP) served as a substrate with Km = 0.64 microM, and Vmax = 25 milliunits/mg and was a competitive inhibitor with respect to ATP. When mant-ATP bound to the enzyme, fluorescence intensity at 440 nm increased. mant-ATP binding of the wild type and the mutant enzymes were compared using the fluorometric method. The Kd values of the T52A and D128N enzymes were infinitely high and could not be measured, while those of the other mutant enzymes increased slightly. These results provide evidence that those amino acids are involved in substrate binding, and they are consistent with the crystallographic data. The results also suggest that Asp-128 does not serve as a nucleophile in catalysis, and since there are no other potential nucleophiles in the active site, we hypothesize that the Fru 6-P,2-kinase reaction is mediated via a transition state stabilization mechanism.

Amino Acids↗
PubMed1995

Quantitative Aspects of the in Vivo Regulation of Pyrophosphate:Fructose-6-Phosphate 1-Phosphotransferase by Fructose-2,6-Bisphosphate.

Pyrophosphate:fructose-6-phosphate 1-phosphotransferase (PFP) was quantified in developing barley (Hordeum vulgare) leaves by immunostaining on western blots using a purified preparation of barley leaf PFP as standard. Fructose-2,6-bisphosphate (Fru-2,6-bisP) was quantified in the same tissues. Depending on age and tissue development, the concentration of PFP varied between 11 and 80 [mu]g PFP protein g-1 fresh weight, which corresponds to 0.09 to 0.65 nmol g-1 fresh weight of each of the [alpha] and [beta] PFP subunits. The level depends primarily on the maturity of the tissue. In the same tissues the concentration of Fru-2,6-bisP varied between 0.07 and 0.46 nmol g-1 fresh weight. Thus, the concentrations of PFP subunits and Fru-2,6-bisP were of the same order of magnitude. In young leaf tissues the concentration of PFP subunits may exceed the concentration of Fru-2,6-bisP. This means that the amount of Fru-2,6-bisP present will be too low to occupy all the allosteric binding sites on PFP even though the concentration of Fru-2,6-bisP exceeds the Ka(Fru-2,6-bisP) by several orders of magnitude. These results are discussed in relation to Fru-2,6-bisP as a regulator of enzyme activities under in vivo conditions.

Journal Article↗
PubMedDate not supplied

[Analysis of allosteric mechanisms of suppressing parasitic recirculation in the futile cycle fructose-6-P--fructose-1,6-P2].

The uncontrollable substrate recirculation in the central futile cycle (FC) in the carbohydrate energy metabolism fructose-6-P (F6P) in equilibrium or formed from fructose-1,6-P2 (FBP), makes it impossible to maintain a stable level of ATP because of its wasteful expenditure in the cycle reactions which are equivalent to the ATPase reaction and also because of the diversion of FBP from glycolytic phosphorylation of ADP. It follows from the analysis of a mathematical model of the carbohydrate energy metabolism that the allosteric inhibition of fructosebisphosphatase (FBPase) by FBP and AMP leads to suppression of the recirculation in the FC and recovery of the ability of glycolysis to stabilize the level of ATP with high accuracy. The allosteric activation of phosphofrucktokinase (PFK) by AMP couples the expenditure of ATP and F6P in the FC with ATP consumption by a load.

Adenosine Monophosphate↗
PubMed1978

[Behavior of various blood constituents (glucose, fructose, insulin, lactate, pyruvate, free fatty acids, inorganic phosphate) and half-life of monosaccharides in the plasma after i.v. infusion of glucose, fructose, galactose and invert sugar solutions in ruminants. 1. Studies in calves and heifers].

Studies into the half-life of monosaccharides as well as into the effects of intravenous infusion of sugar solutions (0.5 g/kg body weight of glucose, fructose, galactose, and invert sugar) on the concentration of various blood components were undertaken with six calves and five heads of young cattle. Half-life values of glucose and galactose were of nearly identical magnitude over the first weeks of age, whereas that of fructose was much longer on account of slower conversion. Infusion of invert sugar solution, therefore, cannot be recommended for calves during the first three weeks of age. Infusion of the above monosaccharides led to more or less strongly pronounced rise of insulin levels in the blood serum. More or less strongly marked pyruvate increase took place following infusion, if the pyruvate concentration in the blood plasma had been low before infusion. Lactate levels in the blood usually underwent little change, whereas the levels of free fatty acids and inorganic phosphate in the blood plasma usually were reduced.

Animals↗
PubMed1993

Significance of the amino terminus of rat testis fructose-6-phosphate, 2-kinase:fructose-2,6-bisphosphatase.

In order to assess the functional properties of the extreme amino-terminal peptide of rat testis fructose-6-phosphate,2-kinase: fructose-2,6-bisphosphatase, 24 and 30 amino acids from the NH2 terminus were deleted using oligonucleotide-directed mutagenesis. The expressed wild-type (WT) and the mutant (Del 24 and Del 30) enzymes were purified to homogeneity. These enzymes were homodimers of subunit M(r) values 54,000, 51,300, and 50,600. Deletion of 1-24 and 1-30 residues resulted in over 70% loss of Fru-6-P,2-kinase activity and 2-fold increase in Fru-2,6-bisphosphatase. KmFru-6-P increased 7.5-fold, but there was no significant change in KmFru-2,6P2 of the phosphatase. These mutant enzymes were more susceptible to protein concentration-dependent dissociation and thermal inactivation. In 2 M urea the kinase and the phosphatase activities of WT increased 17 and 56%, respectively, but no activation of any of the mutant enzymes was observed. At higher urea concentrations, all the enzymes were inactivated. Unfolding in urea, followed with intrinsic tryptophan fluorescence, showed that the unfolding proceeded in at least two stages. Fluorescence intensity at 338 nm of all these enzymes decreased below 2 M urea in which the enzyme activities remained the same (Del 24) or activated (WT). The second stage of unfolding was seen as the maximum shifted to 342 nm in 2-4 M urea, presumably involving dissociation. Complete unfolding above 4 M urea resulted in further shift in fluorescence maximum to 350 nm.

Amino Acid Sequence↗
PubMed1999

On the inhibition of hepatic glycogenolysis by fructose. A 31P-NMR study in perfused rat liver using the fructose analogue 2,5-anhydro-D-mannitol.

Inhibition of hormone-stimulated hepatic glycogenolysis by fructose (Fru) has been attributed to accumulation of the competitive inhibitor Fru1P and/or to the associated depletion of the substrate phosphate (Pi). To evaluate the relative importance of either factor, we used the Fru analogue 2,5-anhydro-D-mannitol (aHMol). This analogue is avidly phosphorylated, traps Pi, and inhibits hormone-stimulated glycogenolysis, but it is not a gluconeogenic substrate, and hence does not confound glycogenolytic glucose production. Livers were continuously perfused with dibutyryl-cAMP (100 microM) to clamp phosphorylase in its fully activated a form. We administered aHMol (3.8 mM), and studied changes in glycogenolysis (glucose, lactate and pyruvate output) and in cytosolic Pi and phosphomonoester (PME), using in situ 31P-NMR spectroscopy (n = 4). Lobes of seven livers perfused outside the magnet were extracted for evaluation, by high-resolution 31P-NMR, of the evolution of aHMol1P and of aHMol(1,6)P2. After addition of aHMol, both glycogenolysis and the NMR Pi signal dropped precipitously, while the PME signal rose continuously and was almost entirely composed of aHMol1P. Inhibition of glycogenolysis in excess of the drop in Pi could be explained by continuing accumulation of aHMol1P. A subsequent block of mitochondrial ATP synthesis by KCN (1 mM) caused a rapid increase of Pi. Despite recovery of Pi to values exceeding control levels, glycogenolysis only recovered partially, attesting to the Pi-dependence of glycogenolysis, but also to inhibition by aHMol phosphorylation products. However, KCN resulted in conversion of the major part of aHMol1P into aHMol(1,6)P2. Residual inhibition of glycogenolysis was due to aHMol1P. Indeed, the subsequent withdrawal of aHMol caused a further gradual decrease in the proportion of aHMol1P (being converted into aHMol(1,6)P2, in the absence of de novo aHMol1P synthesis), and this resulted in a gradual de-inhibition of glycogenolysis, in the absence of marked changes in Pi. Glycogenolytic rates were consistently predicted by a model assuming non-saturated Pi kinetics and competition by aHMol1P exclusively: In conclusion, limited Pi availability and the presence of competitive inhibitors are decisive factors in the control of the in situ catalytic potential of phosphorylase a.

Animals↗
PubMed1984

Fructose 1,6-bisphosphatase in the diagnosis of chronic hepatitis. II. Classification of chronic hepatitis based on fructose 1,6-bisphosphatase and other laboratory data.

The histomorphology of typical liver cell necroses are here correlated with heterotope distributions of enzymes in liver parenchyma. A variety of findings indicate a congruence between gluconeogenetic areas of the liver and the typical pattern of 'piecemeal' necrosis. We therefore propose a diagnostic index based on fructose 1,6-bisphosphatase activity and the data from the clinical laboratory. This index makes it possible to distinguish between chronic persistent and chronic aggressive hepatitis.

Acute Disease↗
PubMed1993

Regulation of rat-kidney cortex fructose-1,6-bisphosphatase activity. I. Effects of fructose-2,6-bisphosphate and divalent cations.

1. The native rat-kidney cortex Fructose-1,6-BPase is differentially regulated by Mg2+ and Mn2+. 2. Mg2+ binding to the enzyme is hyperbolic and large concentrations of the cation are non-inhibitory. 3. Mn2+ produces a 10-fold rise in Vmax higher than Mg2+. [Mn2+]0.5 is much larger than [Mg2+]0.5. At elevated [Mn2+] inhibition is observed. 4. Mg2+ and Mn2+ produce antagonistic effects on the inhibition of the enzyme by high substrate. 5. Fru-2,6-P2 inhibits the enzyme by rising the S0.5 and favouring a sigmoidal kinetics. 6. The inhibition by Fru-2,6-P2 is released by Mg2+ and more powerfully by Mn2+ increasing the I0.5.

Animals↗
PubMed1991

Conformational transition of fructose-1,6-bisphosphatase: structure comparison between the AMP complex (T form) and the fructose 6-phosphate complex (R form).

A structure of the neutral form of fructose-1,6-bisphosphatase complexed with AMP has been determined by the molecular replacement method and refined at a 2.5-A resolution to a crystallographic R factor of 0.169. The root-mean-square errors of the structure from standard geometry are 0.013 A for bond lengths and 2.99 degrees for bond angles. Comparison of the AMP complex with the F6P complex shows that dimer C3-C4 twists about 19 degrees about a molecular 2-fold axis when dimers C1-C2 of the R and T forms of the enzyme are superimposed one another and that a slight shift of about 1 A of the AMP domain partially compensates this twist. The R to T transition of the enzyme does not significantly change the conformation of the F6P-binding site. However, residues at the divalent metal site and the AMP site show significant positional shifts. If these results can be extended to substrate in place of F6P, they suggest that regulation of the enzyme by AMP may occur partly through effects on metal-ion affinity or position. AMP binds to the same sites of the T and R forms, but only half-occupancy was observed in the alkaline R form. Sequential binding of AMP, at least in pairs, is suggested as the unligated R form is converted to the T form. Two possible pathways are suggested for allosteric communication over about 28 A between the AMP site and the active site: one via helices H1, H2, and H3 and another via the eight-stranded beta-sheet.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Monophosphate↗
PubMed1998

Vid24p, a novel protein localized to the fructose-1, 6-bisphosphatase-containing vesicles, regulates targeting of fructose-1,6-bisphosphatase from the vesicles to the vacuole for degradation.

Glucose regulates the degradation of the key gluconeogenic enzyme, fructose-1,6-bisphosphatase (FBPase), in Saccharomyces cerevisiae. FBPase is targeted from the cytosol to a novel type of vesicle, and then to the vacuole for degradation when yeast cells are transferred from medium containing poor carbon sources to fresh glucose. To identify proteins involved in the FBPase degradation pathway, we cloned our first VID (vacuolar import and degradation) gene. The VID24 gene was identified by complementation of the FBPase degradation defect of the vid24-1 mutant. Vid24p is a novel protein of 41 kD and is synthesized in response to glucose. Vid24p is localized to the FBPase-containing vesicles as a peripheral membrane protein. In the absence of functional Vid24p, FBPase accumulates in the vesicles and fails to move to the vacuole, suggesting that Vid24p regulates FBPase targeting from the vesicles to the vacuole. FBPase sequestration into the vesicles is not affected in the vid24-1 mutant, indicating that Vid24p acts after FBPase sequestration into the vesicles has occurred. Vid24p is the first protein identified that marks the FBPase-containing vesicles and plays a critical role in delivering FBPase from the vesicles to the vacuole for degradation.

Amino Acid Sequence↗
PubMed1994

Catabolite inactivation of heterologous fructose-1,6-bisphosphatases and fructose-1,6-bisphosphatase-beta-galactosidase fusion proteins in Saccharomyces cerevisiae.

Fructose-1,6-bisphosphatase (FruP2ase) from Saccharomyces cerevisiae is rapidly inactivated upon addition of glucose to a culture growing on non-sugar carbon sources. Under the same conditions the FruP2ases from Schizosaccharomyces pombe or Escherichia coli expressed in S. cerevisiae were not affected. A chimaeric protein containing the first 178 amino acids from the N-terminal half of S. cerevisiae FruP2ase fused to E. coli beta-galactosidase was susceptible to catabolite inactivation. Elimination of a putative destruction box, RAELVNLVG ... KK .... K., beginning at amino acid 60 did not prevent catabolite inactivation. Similarly a change of the vacuole-targeting sequence QKKLD, amino acids 80-84, to QKNSD did not affect significantly the course of inactivation of beta-galactosidase. A fusion protein carrying only the first 138 amino acids from FruP2ase was inactivated at a higher rate than the one carrying the first 178, suggesting the existence of a protective region between amino acids 138 and 178. A fusion protein carrying the first 81 amino acids from FruP2ase was inactivated by glucose at a similar rate to the one carrying the 178 amino acids, but one with only the first 18 amino acids was resistant to catabolite inactivation. Inactivation of FruP2ase in mutants ubr1 that lack a protein required for ubiquitin-dependent proteolysis, or pra1 that lack vacuolar protease A, proceeded as in a wild type. Our results suggest that at least two domains of FruP2ase may mark beta-galactosidase for catabolite inactivation and that FruP2ase can be inactivated by a mechanism independent of transfer to the vacuole.

Amino Acid Sequence↗
PubMed2006

Catabolism of 1,5-anhydro-D-fructose in Sinorhizobium morelense S-30.7.5: discovery, characterization, and overexpression of a new 1,5-anhydro-D-fructose reductase and its application in sugar analysis and rare sugar synthesis.

The bacterium Sinorhizobium morelense S-30.7.5 was isolated by a microbial screening using the sugar 1,5-anhydro-D-fructose (AF) as the sole carbon source. This strain metabolized AF by a novel pathway involving its reduction to 1,5-anhydro-D-mannitol (AM) and the further conversion of AM to D-mannose by C-1 oxygenation. Growth studies showed that the AF metabolizing capability is not confined to S. morelense S-30.7.5 but is a more common feature among the Rhizobiaceae. The AF reducing enzyme was purified and characterized as a new NADPH-dependent monomeric reductase (AFR, EC 1.1.1.-) of 35.1 kDa. It catalyzed the stereoselective reduction of AF to AM and also the conversion of a number of 2-keto aldoses (osones) to the corresponding manno-configurated aldoses. In contrast, common aldoses and ketoses, as well as nonsugar aldehydes and ketones, were not reduced. A database search using the N-terminal AFR sequence retrieved a putative 35-kDa oxidoreductase encoded by the open reading frame Smc04400 localized on the chromosome of Sinorhizobium meliloti 1021. Based on sequence information for this locus, the afr gene was cloned from S. morelense S-30.7.5 and overexpressed in Escherichia coli. In addition to the oxidoreductase of S. meliloti 1021, AFR showed high sequence similarities to putative oxidoreductases of Mesorhizobium loti, Brucella suis, and B. melitensis but not to any oxidoreductase with known functions. AFR could be assigned to the GFO/IDH/MocA family on the basis of highly conserved common structural features. His6-tagged AFR was used to demonstrate the utility of this enzyme for AF analysis and synthesis of AM, as well as related derivatives.

Amino Acid Sequence↗
PubMed1982

In-vivo comparison of the adsorption capacity of "superactive charcoal" and fructose with activated charcoal and fructose.

This study was undertaken to assess the in-vivo capacity of two activated charcoal products to adsorb aspirin after its ingestion by seven healthy volunteers. The two products, Norit-A and Super-Sorb, were combined with fructose solution and administered after the subjects ingested 975 mg of aspirin. Urinary excretion of salicylates was measured during both charcoal administration phases and after ingestion of aspirin alone in all subjects. Results showed statistically significant differences in salicylate excretion between all phases. Super-Sorb ("superactive charcoal") adsorbed almost twice (1.7) as much as aspirin as the regular activated charcoal, Norit-A. Super-Sorb therefore has a greater in-vivo adsorption capacity for aspirin and should be a more effective antidote in poisonings with this drug.

Adjuvants, Pharmaceutic↗
PubMed1984

Measurement of the rate of substrate cycling between fructose 6-phosphate and fructose 1,6-bisphosphate in skeletal muscle by using a single-isotope technique.

The effects of several agents on the rates of the fructose 6-phosphate/fructose 1,6-bisphosphate substrate cycle were measured in incubated epitrochlearis muscles of the rat by monitoring the transfer of radiolabel from [6-14C]glucose to the 1-position of glucose residues in glycogen. The cycling rates observed were almost identical with those previously obtained by using the well-established dual-isotope technique. In particular, it was found that the beta-adrenoceptor agonist isoprenaline increased the cycling rate about 12-fold.

Animals↗
PubMed1991

Regulation of fructose-2,6-bisphosphate content in mantle tissue of the sea mussel Mytilus galloprovincialis Lmk. Regulation of fructose-2,6-bisphosphatase activity.

Fructose-2,6-bisphosphatase (FBPase-2) from the mantle tissue of the mussel Mytilus galloprovincialis shows a hyperbolic kinetic with a Km value (0.40 mM) for its substrate, that suggest that the "in vivo" Fru-2,6-P2 concentration is not a limiting factor for activity. The enzyme possesses an optimum pH for activity between 6 and 7 units, similar to the reached in mussel mantle during physiological hypoxia. The modulation of activity by the pH, and in addition, the positive effect of ATP are in keeping with the little decrease in concentration of the Fru-2,6-P2 that occurs during the first hour of hypoxia due to the valve closure.

Adenosine Triphosphate↗