Solution structure of 5-keto-D-fructose: relevance to the specificity of hexose kinases.

5-Keto-D-fructose (5KF) is isolated from cultures of Gluconobacter cerinus growing on D-fructose as the sole carbon source. 5KF is a substrate for hexokinase, fructokinase, and several polyol dehydrogenases. 1H and 13C nuclear magnetic resonance studies show that 5KF exists in different forms in anhydrous dimethyl-d6 sulfoxide and D2O. In dimethyl-d6 sulfoxide, 5KF exists as a spirane dimer with linked furanose and pyranose rings, similar to the structure reported for crystalline 5KF [Hassen, L., Hordvik, A., & Hove, R. (1976) J. Chem. Soc., Chem. Commun., 572-. In D2O, 5KF exists predominantly (greater than 95%) in a beta-pyranose form with the 5-keto group hydrated to form a gem-diol. 13C--1H coupling patterns, 13C relaxation measurements, and 13C deuterium-induced differential isotope shifts confirm this structure of 5KF. The phosphorylation of 5KF by fructokinase can be accounted for by an approximately 2% proportion of the beta-furanose form in solution at 25 degrees C. Both the beta-pyranose and beta-furanose forms of 5KF are proposed to be substrates for yeast hexokinase.