Des-1-25-fructose-1,6-bisphosphatase, a nonallosteric derivative produced by trypsin treatment of the native protein.
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Abstract Limited tryptic digestion of pig kidney fructose-1,6-bisphosphatase in the presence of magnesium ions results in the formation of an active enzyme derivative which is no longer inhibited by the allosteric effector AMP. The presence of AMP during incubation of fructose-1,6-bisphosphatase with trypsin protects against the loss of AMP inhibition. By contrast, the presence of the nonhydrolyzable substrate analog fructose 2,6-bisphosphate accelerates the rate of formation of that form of fructose-1,6-bisphosphatase which is insensitive to AMP inhibition. Sodium dodecyl sulfate-polyacrylamide electrophoresis of samples taken during trypsin treatment shows that the loss of AMP inhibition parallels the conversion of the native 36,500 molecular weight fructose-1,6-bisphosphatase subunit into a 34,000 molecular weight species. Automated Edman degradation of trypsin-treated fructose-1,6-bisphosphatase following gel filtration shows a single sequence beginning at Gly-26 in the original enzyme, but no changes in the COOH-terminal region of fructose-1,6-bisphosphatase. Thus, the proteolytic product has been characterized as A comparison of the kinetic properties of control enzyme and des-1-25-fructose-1,6-bisphosphatase reveals some differences in properties (pH optimum, Ka for Mg2+, K+ activation, inhibition by fructose 2,6-bisphosphate) between the two enzymes, but none is so striking as the complete loss of AMP sensitivity shown by des-1-25-fructose-1,6-bisphosphatase. The loss of AMP inhibition is due to the loss of AMP-binding capacity, but it is not known at this stage whether residues of the AMP site are present in the 25-amino acid NH2-terminal region or the removal of this region leads to a conformational change that abolishes the function of an AMP site located elsewhere in the molecule.Keywords:
Fructose 1,6-bisphosphatase
Aldolase B
A sensitive new method in which D-[U-14C]fructose-1-phosphate is used for fructose-bisphosphate aldolase (EC 2.1.2.13) assay is described. The radioactive fructose-1-phosphate compound was prepared from [U-14C]fructose by use of partly purified fructokinase (EC 2.7.1.4). With this method we measured normal values for aldolase in human liver (2.4-10.0 nmol/min per mg of protein), kidney (3.6-3.8), and intestine (4.2-10.0) as well as Km values for fructose-1-phosphate (approximately 1.0-2.2 mmol/L). In patients with hereditary fructose intolerance the aldolase activity in liver and intestine was less than 10% of normal values. The Lineweaver-Burk plots for data from patients with hereditary fructose intolerance were hyperbolic, indicating a structural alteration in the enzyme.
Aldolase B
Fructolysis
Fructokinase
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Mutants of Escherichia coli impaired in fructose 1-phosphate kinase activity (Fpk) accumulate fructose 1-phosphate from fructose, which arrests their growth. Phenotypic revertants to fructose tolerance have lost either the histidine-containing protein (HPr) or enzyme I of the phosphoenolpyruvate phosphotransferase system ( ctr ), and consequently utilize neither fructose nor glucose, or an enzyme II specific for the uptake of fructose and its concomitant phosphorylation to fructose 1-phosphate (PtsF). However, PtsF — -mutants can still grow on high concentrations ( > 2 mM) of fructose, and take up this sugar via a low-affinity enzyme II designated PtsX that effects its uptake and phosphorylation to fructose 6-phosphate. Mutants of the Hfr-strain KL16 were isolated that lacked PtsF, or PtsF and PtsX, activities. The consequence to, and rôle of these functions in, the uptake and metabolism of fructose are described.
PEP group translocation
Aldolase B
Fructolysis
Fructose 1,6-bisphosphatase
Phosphotransferases
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Understanding how glucose metabolism is finely regulated at molecular and cellular levels in the liver is critical for knowing its relationship to related pathologies, such as diabetes. In order to gain insight into the regulation of glucose metabolism, we studied the liver-expressed isoforms aldolase B and fructose-1,6-bisphosphatase-1 (FBPase-1), key enzymes in gluconeogenesis, analysing their cellular localization in hepatocytes under different metabolic conditions and their protein-protein interaction in vitro and in vivo. We observed that glucose, insulin, glucagon and adrenaline differentially modulate the intracellular distribution of aldolase B and FBPase-1. Interestingly, the in vitro protein-protein interaction analysis between aldolase B and FBPase-1 showed a specific and regulable interaction between them, whereas aldolase A (muscle isozyme) and FBPase-1 showed no interaction. The affinity of the aldolase B and FBPase-1 complex was modulated by intermediate metabolites, but only in the presence of K(+). We observed a decreased association constant in the presence of adenosine monophosphate, fructose-2,6-bisphosphate, fructose-6-phosphate and inhibitory concentrations of fructose-1,6-bisphosphate. Conversely, the association constant of the complex increased in the presence of dihydroxyacetone phosphate (DHAP) and non-inhibitory concentrations of fructose-1,6-bisphosphate. Notably, in vivo FRET studies confirmed the interaction between aldolase B and FBPase-1. Also, the co-expression of aldolase B and FBPase-1 in cultured cells suggested that FBPase-1 guides the cellular localization of aldolase B. Our results provide further evidence that metabolic conditions modulate aldolase B and FBPase-1 activity at the cellular level through the regulation of their interaction, suggesting that their association confers a catalytic advantage for both enzymes.
Fructose 1,6-bisphosphatase
Aldolase B
Phosphofructokinase 2
Gluconeogenesis
Glucokinase
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Aldolase B
Fructolysis
Gluconeogenesis
Fructose 1,6-bisphosphatase
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Abstract Fructose is an important macronutrient and fructose esters participate critically in intermediary metabolism; thus, inherited defects of enzymes involved in handling of dietary fructose may have profound metabolic effects. Examples are essential fructosuria, hereditary fructose intolerance and fructose‐1,6‐bisphosphatase deficiency.
Fructose 1,6-bisphosphatase
Fructolysis
Intermediary Metabolism
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Aldolase B
Fructolysis
Phosphofructokinase 2
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A selection system for yeast cells expressing mutated invertase (EC 3.2.1.26, SUC2 ) with altered fructotransferase activity was developed based on the survival of a fructose-intolerant strain in the presence of suitable acceptor substrates and sucrose. Cells of such a strain expressing a wild-type hydrolase activity will not grow due to the release of free fructose from sucrose. Cells expressing an inactive invertase mutant will not grow since they cannot cleave the sucrose, the sole carbon source. Only cells expressing sucrose fructosyl-transferase activity will thrive, growing on the released glucose, the fructosyl moiety not being released. A strain of Saccharomyces cerevisiae was engineered to be intolerant of the presence of fructose in its growth media. This was achieved by inducing a condition in yeast similar to liver cells of humans suffering from hereditary fructose intolerance (MIM 22960). This disorder results from a deficiency of aldolase B (EC 4.1.2.13), and phosphorylation of fructose by ketohexokinase (EC 2.7.1.3) results in an accumulation of fructose 1-phosphate, with a consequent depletion of cytoplasmic phosphate and ATP. Thus, cells in which ketohexokinase phosphorylates fructose, but which lack aldolase B, are intolerant of fructose. Yeast possess neither of these enzymes, and so expression of ketohexokinase in yeast would result in fructose-intolerance. A strain of yeast, for ketohexokinase expression, was initially bred to be unable to metabolize sucrose or fructose, yet remain capable of utilizing glucose, as well as lacking non-specific phosphatases, to prevent remobilization of sequestered fructose 1-phosphate. Rat liver ketohexokinase was purified to heterogeneity, and the partial amino acid sequence subsequently generated exploited to amplify a region of the ketohexokinase cDNA by PCR. This was used to probe a cDNA library, yielding clones encoding the entire ketohexokinase coding region. This was cloned into pMA91 , and subsequent expression in yeast resulted in a strain intolerant of fructose in its growth medium, although still capable of growing on glucose. In order to produce a stable fructose-intolerant selection strain, a vector ( pIAD l) was constructed that allowed multiple integration of an expression cassette containing ketohexokinase cDNA into the rDNA locus of yeast chromosome XII. Expression of wild type invertase from the episomal plasmid p IAD 3 in this strain resulted in sucrose-intolerance. A preliminary programme of mutagenesis of the SUC2 gene yielded eight libraries of about one hundred clones each. None of these contained any mutants showing solely sucrose fructosyl-transferase activity, although this system would clearly provide an ideal selection for such mutants from a much larger library.
Aldolase B
Fructokinase
Fructose 1,6-bisphosphatase
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Abstract. The metabolism of fructose by the small intestine can be analyzed in terms of the following scheme: 1) hydrolysis of fructose containing saccharides especially sucrose; 2) movement of fructose into the intestinal cell; 3) transformation of fructose into glycolytic metabolic intermediates; 4) formation of fructose from glucose via sorbitol; 5) adaptive regulation of fructose metabolizing enzymes; 6) adaptive responses of other enzymes to fructose. The hydrolysis of sucrose is dependent upon the brush border enzyme sucrase which shows an adaptive response to sucrose diets. The entrance of fructose into the small intestine and the intermediary metabolism of fructose is reviewed. Fructose metabolizing enzymes, fructokinase and fructose‐1‐phosphate aldolase, are regulated by the presence or absence of fructose, folic acid and drugs. Fructose causes adaptive changes in small intestine glycolytic enzymes and decreases the gluconeogenic enzyme fructose‐1,6‐diphosphatase. Actinomycin D inhibits the adaptive effect of fructose on glycolytic enzymes which suggests that fructose acts via the protein synthetic mechanism.
Fructokinase
Fructolysis
Fructose 1,6-bisphosphatase
Aldolase B
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We observed that glucose, insulin, glucagon and adrenaline differentially modulate the intracellular distribution of aldolase-B and FBPase-1. Interestingly, the in vitro protein-protein interaction analysis between aldolase-B and FBPase-1 showed a specific and regulatable interaction between them, whereas aldolase-A (muscle isozyme) and FBPase-1 showed no interaction. The affinity of the aldolase-B and FBPase-1 complex was modulated by intermediate metabolites, but only in the presence of K. We observed a decreased association constant in the presence of AMP, fructose-2,6-bisphosphate, fructose-6-phosphate and inhibitory concentrations of fructose-1,6-bisphosphate. Conversely, the association constant of the complex increased in the presence of dihydroxyacetone phosphate and non-inhibitory concentrations of fructose-1,6-bisphosphate. Notably, in vivo FRET studies confirmed the interaction between aldolase-B and FBPase-1. Also, the co-expression of aldolase-B and FBPase-1 in cultured cells suggested that FBPase-1 guides the cellular localization of aldolaseB.
Aldolase B
Fructose 1,6-bisphosphatase
Phosphofructokinase 2
Gluconeogenesis
Fructose 2,6-bisphosphate
Fructolysis
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An inborn deficiency in the ability of aldolase B to split fructose 1-phosphate is found in humans with hereditary fructose intolerance (HFI). A stable isotope procedure to elucidate the mechanism of conversion of fructose to glucose in normal children and in HFI children has been developed. A constant infusion of D-[U-13C]fructose was given nasogastrically to control and to HFI children. Hepatic fructose conversion to glucose was estimated by examination of 13C NMR spectra of plasma glucose. The conversion parameters in the control and HFI children were estimated on the basis of doublet/singlet values of the plasma beta-glucose C-1 splitting pattern as a function of the rate of fructose infusion (0.26-0.5 mg/kg per min). Significantly lower values (approximately 3-fold) for fructose conversion to glucose were obtained for the HFI patients as compared to the controls. A quantitative determination of the metabolic pathways of fructose conversion to glucose was derived from 13C NMR measurement of plasma [13C]glucose isotopomer populations. The finding of isotopomer populations of three adjacent 13C atoms at glucose C-4 (13C3-13C4-13C5) suggests that there is a direct pathway from fructose, by-passing fructose-1-phosphate aldolase, to fructose 1,6-bisphosphate. The metabolism of fructose by fructose-1-phosphate aldolase activity accounts for only approximately 50% of the total amount of hepatic fructose conversion to glucose. It is suggested that phosphorylation of fructose 1-phosphate to fructose 1,6-bisphosphate by 1-phosphofructokinase occurs in human liver (and intestine) when fructose is administered nasogastrically; 47% and 27% of the total fructose conversion to glucose in controls and in HFI children, respectively, takes place by way of this pathway. In view of the marked decline by 67% in synthesis of glucose from fructose in HFI subjects found in this study, the extent of [13C]glucose formation from a "trace" amount (approximately 20 mg/kg) of [U-13C]fructose infused into the patient can be used as a safe and noninvasive diagnostic test for inherent faulty fructose metabolism.
Aldolase B
Fructolysis
Isotopomers
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