The substrate and anomeric specificity of fructokinase.

Fructokinase from beef liver is shown to be specific for the /3-furanose anomer of D-fructose (and the LY anomer of Lsorbose) by showing that certain 2,5-anhydroalditols are substrates, while other 2,5-anhydro compounds, 2,6-anhydro-D-mannitol, and 2,6-anhydro-D-glucitol are not. Km and Ir,,, values relative to D-fructose at pH 7.5, 25”, 4 mu MgATP are 2,5-anhydro-D-mannitol (1.7 mu, 1.97); 2,5anhydro-D-glucitol (5.9 mu, 1.33, phosphorylated in position 6); 2,5-anhydro-D-lyxitol (67 mk& 0.19); 2,5-anhydro-Dmannose (1.5 mu, 0.50). 2,5-Anhydro-D-xylitol inhibits (Ki, 40 mu), while 2,5-anhydro-L-iditol and cis 2,5-bis(hydroxymethyl)tetrahydrofuran are neither inhibitors nor substrates. Based on these results and other published data, the substrate specificity of fructokinase is for a tetrahydrofuran ring with /3-D-(or a-~-) configuration at position 2, Lconfiguration at postion 3, and either Dor L-configuration at positions 4 and 5. With yeast hexokinase the Km and V,., values relative to D-fructose of several anhydro compounds at pH 7.5, 25’, 2 m&z MgATP, are 2,5-anhydro-D-mannitol (6.3 mu, 0.52); 2,5-anhydro-D-glucitol (47 m& 0.37) ; 2,5-anhydro-D-mannose (0.31 mM, 0.37).