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PubMed1977

Bovine liver fructokinase: purification and kinetic properties.

Fructokinase from beef liver has been purified 2300-fold by acid and heat treatment, ammonium sulfate fractionation, and chromatography on Sephadex G-100, DEAE- and CM-cellulose. The purified enzyme is homogeneous by all criteria examined, has a molecular weight of 56 000, and is a dimer of equal molecular weight subunits. The isoelectric point is 5.7. The Michaelis constant for activation by K+ is 15 mM, and the enzyme is also activated by Na+, Rb+, Cs+, NH4+, and TL+. The kinetic mechanism has been determined at pH 7.0, 25 degrees C. The initial velocity, product, and dead-end inhibition patterns for CrATP, CrADP, and 1-deoxy-D-fructose are consistent with a random kinetic mechanism with the formation of two dead-end complexes. Substrates for fructokinase include: D-fructose, L-sorbose, D-tagatose, D-psicose, D-xylulose, L-ribulose, D-sedoheptulose, L-galactoheptulose, D-mannoheptulose, 5-keto-D-fructose, D-ribose, 2,5-anhydro-D-mannitol, 2,5-anhydro-D-glucitol, 2,5-anhydro-D-mannose, 2,5-anhydro-D-lyxito.l, and D-ribono-gamma-lactone. 5-Thio-D-fructose was not a substate, but was a competitive inhibitor vs. D-fructose. Thus the minimum molecular for substrate activity seems to be (2R)-2-hydroxy-methyl-3,4-dihydroxytetrahydrofuran. The configuration of the substituents at carbons 3, 4, and 5 appears not to be critical, but the hydroxymethyl group must have the configuration corresponding to beta-D-(or alpha-L-) keto sugars. The anomeric hydroxyl on carbon 2 is not required (although it contributes to binding), and a wide variety of groups may be present at carbon 5.

Animals↗
PubMed1998

Tomato fructokinases exhibit differential expression and substrate regulation

Two divergent genes encoding fructokinase, Frk1 and Frk2, have been previously shown to be expressed in tomato (Lycopersicon esculentum L.) and have now been further characterized with regard to their spatial expression and the enzymic properties of the encoded proteins. Frk1 and Frk2 mRNA levels were coordinately induced by exogenous sugar, indicating that both belong to the growing class of sugar-regulated genes. However, in situ hybridization indicated that Frk1 and Frk2 were expressed in a spatially distinct manner, with Frk2 mRNA primarily localized in cells of the fruit pericarp, which store starch, and Frk1 mRNA distributed ubiquitously in pericarp tissue. To evaluate the biochemical characteristics of the products of the Frk1 and Frk2 genes, each cDNA was expressed in a mutant yeast (Saccharomyces cerevisiae) line defective in hexose phosphorylation and unable to grow on glucose or fructose (Fru). Both Frk1 and Frk2 proteins expressed in yeast conferred the ability to grow on Fru and exhibited fructokinase activity in vitro. Although both Frk1 and Frk2 both utilized Fru as a substrate, only Frk2 activity was inhibited at high Fru concentrations. These results indicate that Frk2 can be distinguished from Frk1 by its sensitivity to substrate inhibition and by its temporal and spatial pattern of expression, which suggests that it plays a primary role in plant cells specialized for starch storage.

Journal Article↗
PubMed1990

Fructokinases from developing maize kernels differ in their specificity for nucleoside triphosphates.

A new form of fructokinase has been identified from developing maize (Zea mays L.) kernels that utilizes CTP, UTP, and GTP from four to eight times more effectively than ATP at nonlimiting concentrations. Ten millimolar dithiothreitol was necessary to stabilize activity. A second form of fructokinase was nonspecific for nucleoside triphosphate whereas a third form was fairly specific for ATP.

Journal Article↗
PubMed2004

Molecular and biochemical characterization of a fructose-6-phosphate-forming and ATP-dependent fructokinase of the hyperthermophilic archaeon Thermococcus litoralis.

Close to an operon encoding an ABC transporter for maltose and trehalose, Thermococcus litoralis contains a gene whose encoded sequence showed similarity to sugar kinases. We cloned this gene, now called frk, and expressed it as a C-terminal His-tag version in Escherichia coli. We purified the recombinant protein, identified it as an ATP-dependent and fructose-6-phosphate-forming fructokinase (Frk) and determined its biochemical properties. At its optimal temperature of 80 degrees C, the apparent Km and Vmax values of Frk were 2.3 mM and 730 U/mg protein for fructose at saturating ATP concentration, and 0.81 mM and 920 U/mg protein for ATP at saturating fructose concentration. The enzyme did not lose activity at 80 degrees C for 4 h. Under denaturating conditions in SDS-PAGE, it exhibited a molecular mass of 35 kDa. Gel-filtration chromatography revealed a molecular mass of 58 kDa, indicating a dimer under nondenaturating, in vitro conditions.

Adenosine Triphosphate↗
PubMed1984

Kinetic studies of fructokinase I of pea seeds.

Fructokinase I of pea seeds has been purified to homogeneity and the enzyme shown to be monomeric, with a molecular weight of 72,000 +/- 4000. The reaction mechanism was investigated by means of initial velocity studies. Both substrates inhibited the enzyme; the inhibition caused by MgATP was linear-uncompetitive with respect to fructose whereas that caused by D-fructose was hyperbolic-noncompetitive against MgATP. The product D-fructose 6-phosphate caused hyperbolic-noncompetitive inhibition with respect to both substrates. MgADP caused noncompetitive inhibition, which gave intercept and slope replots that were linear with D-fructose but hyperbolic with MgATP. Free Mg2+ caused linear-uncompetitive inhibition when either substrate was varied. L-Sorbose and beta, gamma-methyleneadenosine 5'-triphosphate were used as analogs of D-fructose and MgATP, respectively. Inhibition experiments using these compounds indicated that substrate addition was steady-state ordered, with MgATP adding first. The product inhibition experiments were found to be consistent with a steady-state random release of products. The substrate inhibition caused by MgATP was most likely due to the formation of an enzyme-MgATP-product dead-end complex, whereas that caused by D-fructose was due to alternative pathways in the reaction mechanism. The inhibition caused by Mg2+ can be explained in terms of a dead-end complex with either a central complex or an enzyme-product complex.

Adenosine Diphosphate↗
PubMed1996

Cloning of human adenosine kinase cDNA: sequence similarity to microbial ribokinases and fructokinases.

Adenosine kinase catalyzes the phosphorylation of adenosine to AMP and hence is a potentially important regulator of extracellular adenosine concentrations. Despite extensive characterization of the kinetic properties of the enzyme, its primary structure has never been elucidated. Full-length cDNA clones encoding catalytically active adenosine kinase were obtained from lymphocyte, placental, and liver cDNA libraries. Corresponding mRNA species of 1.3 and 1.8 kb were noted on Northern blots of all tissues examined and were attributable to alternative polyadenylylation sites at the 3' end of the gene. The encoding protein consists of 345 amino acids with a calculated molecular size of 38.7 kDa and does not contain any sequence similarities to other well-characterized mammalian nucleoside kinases, setting it apart from this family of structurally and functionally related proteins. In contrast, two regions were identified with significant sequence identity to microbial ribokinase and fructokinases and a bacterial inosine/guanosine kinase. Thus, adenosine kinase is a structurally distinct mammalian nucleoside kinase that appears to be akin to sugar kinases of microbial origin.

Adenosine Kinase↗
PubMed1994

Molecular basis of essential fructosuria: molecular cloning and mutational analysis of human ketohexokinase (fructokinase).

Essential fructosuria is one of the oldest known inborn errors of metabolism. It is a benign condition which is believed to result from deficiency of hepatic fructokinase (ketohexokinase, KHK, E.C.2.7.1.3). This enzyme catalyses the first step of metabolism of dietary fructose, conversion of fructose to fructose-1-phosphate. Despite the early recognition of this disorder, the primary structure of human KHK and the molecular basis of essential fructosuria have not been previously defined. In this report, the isolation and sequencing of full-length cDNA clones encoding human ketohexokinase are described. Alternative mRNA species and alternative KHK isozymes are produced by alternative polyadenylation and splicing of the KHK gene. The KHK proteins show a high level of sequence conservation relative to rat KHK. Direct evidence that mutation of the KHK structural gene is the cause of essential fructosuria was also obtained. In a well-characterized family, in which three of eight siblings have fructosuria, all affected individuals are compound heterozygotes for two mutations Gly40Arg and Ala43Thr. Both mutations result from G-->A transitions, and each alters the same conserved region of the KHK protein. Neither mutation was seen in a sample of 52 unrelated control individuals. An additional conservative amino acid change (Val49IIe) was present on the KHK allele bearing Ala43Thr.

Alternative Splicing↗
PubMed1991

Molecular analysis of two fructokinases involved in sucrose metabolism of enteric bacteria.

Sucrose-positive derivatives of Escherichia coli K-12, containing the plasmid pUR400, and of Klebsiella pneumoniae hydrolyse intracellular sucrose 6-phosphate by means of an invertase into D-glucose 6-phosphate and free D-fructose. The latter is phosphorylated by an ATP-dependent fructokinase (gene scrK of an scr regulon) to D-fructose 6-phosphate. The lack of ScrK does not cause any visible phenotype in wild-type strains of both organisms. Using genes and enzymes normally involved in D-arabinitol metabolism from E. coli C and K. pneumoniae, derivatives of E. coli K-12 were constructed which allowed the identification of scrK mutations on conventional indicator plates. Cloning and sequencing of scrK from sucrose plasmid pUR400 and from the chromosome of K. pneumoniae revealed an open reading frame of 924 bp in both cases--the equivalent of a peptide containing 307 amino acid residues (Mr 39 and 34 kDa, respectively, on sodium dodecyl sulphate gels). The sequences showed overall identity among each other (69% identical residues) and to a kinase from Vibrio alginolyticus (57%) also involved in sucrose metabolism, lower overall identity (39%) to a D-ribose-kinase from E. coli, and local similarity to prokaryotic, and eukaryotic phosphofructokinases at the putative ATP-binding sites.

Amino Acid Sequence↗
PubMed1975

Kinetic properties and related changes of molecular weight in a fructokinase from Streptomyces violaceoruber.

1. A study of the initial reaction rates at variable substrate concentrations and of the molecular weight of the enzyme in the presence of different effectors, has been carried out using fructokinase (ATP: fructose 6-phosphotransferase, EC 2.7.1.4) from Streptomyces violaceoruber. 2. Saturation curves for MgATP or CoATP are sigmoidal and they change to hyperbolic in the presence of 10 mM Mg2+ or Co2+ in excess over the nucleoside triphosphate. 3. Saturation cuvves for fructose show intermediary plateaux at high (but not at low) concentrations of ATP or Mg2+. 4. The molecular weight of the enzyme in the presence of high concentrations of MgATP is 80 000. In the presence of fructose, and/or Mg2+, the molecular weight is 20 000. 5. The effects of MgADP, uncomplexed ADP or ATP, and low concentrations of detergent on the kinetics have been studied. The results are interpreted as showing the existence of cooperative effects.

Adenosine Triphosphate↗
PubMed1977

Determination of the rate-limiting steps and chemical mechanism of fructokinase by isotope exchange, isotope partitioning, and pH studies.

Isotope exchange studies show that beef liver fructokinase has a random kinetic mechanism in which release of fructose from the enzyme is slower than that catalytic reaction. The stickiness of fructose in the presence of MgATP is confirmed by isotope partition studies, which show it to be released 0.53 times as fast as V1/Et in the presence, and 80--130 times as fast in the absence of MgATP. Fructose-1-P release from it binary complex is not at all rate limiting in the forward direction since no exchange of MgADP back into MgATP could be observed during the forward reaction. Failure to find any isotope effect by the equilibrium perturbation method with [1-18O]fructose (upper limit, 1.003, shows that P--O bond cleavage or formation is not rate limiting. The pH profiles for the forward reaction show a group (probably carboxyl with pK 5.7-6.0 and deltaHion = 0) that must be ionized and a group (perhaps lysine, with pK 9--10, and deltaHion 5-9 kcal/mol) which must be protonated for activity. The profile for the back reaction shows only a group with pK 5.5--6 that must be protonated for activity. A chemical mechanism is proposed in which a carboxyl group on the enzyme accepts a proton from the 1-hydroxyl of fructose during the forward reaction and donates it back during the reverse reaction.

Adenosine Triphosphate↗