Publications

• Bovine liver fructokinase: purification and kinetic properties.

  Raushel F.M., Cleland W.W.

  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 ... >> More

  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. << Less

  Biochemistry 16:2169-2175(1977) [PubMed] [EuropePMC]

• Properties of normal and mutant recombinant human ketohexokinases and implications for the pathogenesis of essential fructosuria.

  Asipu A., Hayward B.E., O'Reilly J., Bonthron D.T.

  Alternative splicing of the ketohexokinase (fructokinase) gene generates a "central" predominantly hepatic isoform (ketohexokinase-C) and a more widely distributed ketohexokinase-A. Only the abundant hepatic isoform is known to possess activity, and no function is defined for the lower levels of k ... >> More

  Alternative splicing of the ketohexokinase (fructokinase) gene generates a "central" predominantly hepatic isoform (ketohexokinase-C) and a more widely distributed ketohexokinase-A. Only the abundant hepatic isoform is known to possess activity, and no function is defined for the lower levels of ketohexokinase-A in peripheral tissues. Hepatic ketohexokinase deficiency causes the benign disorder essential fructosuria. The molecular basis of this has been defined in one family (compound heterozygosity for mutations Gly40Arg and Ala43Thr). Here we show that both ketohexokinase isoforms are indeed active. Ketohexokinase-A has much poorer substrate affinity than ketohexokinase-C for fructose but is considerably more thermostable. The Gly40Arg mutation seems null, rendering both ketohexokinase-A and ketohexokinase-C inactive and largely insoluble. The Ala43Thr mutant retains activity, but this mutation decreases the thermal stability of both ketohexokinase-A and ketohexokinase-C. At physiologic temperature, this results in significant loss of ketohexokinase-C activity but not of ketohexokinase-A. Affected individuals who carry both mutations therefore probably have a selective deficiency of hepatic ketohexokinase, with peripheral ketohexokinase-A being preserved. These findings raise the possibility that ketohexokinase-A serves an unknown physiologic function that remains intact in essential fructosuria. Further mutation analysis in this rare disorder could illuminate the question of whether ketohexokinase-A activity is, unlike that of ketohexokinase-C, physiologically indispensable. << Less

  Diabetes 52:2426-2432(2003) [PubMed] [EuropePMC]