Hexose-6-kinases in germinating honey locust cotyledons: Substrate specificity of d-fructo-6-kinase

Extracts of the cotyledons of germinated honey locust ( Gleditsia triacanthos) seeds, which contain galactomannan as a reserve polysaccharide in the endosperm, were fractionated by chromatography and the fractions examined for the presence of a specific manno-6-kinase which could phosphorylate the d-mannose released by hydrolysis of galactomannan. One particulate hexokinase (the major hexose-6-kinase fraction) and two soluble hexokinase fractions (the minor portion), as well as a soluble fructo-6-kinase fraction, were initially separated. From chromatography, electrophoresis and kinetic studies, no evidence for a specific manno-kinase was obtained. This and the level and kinetic behaviour of the particulate hexokinase implicated it as the enzyme catalysing the phosphorylation of released d-mannose. The fructo-kinase activity was further separated into three fractions. Kinetic studies on one of these with native and synthetic substrates indicated that the structural requirements for the monosaccharide substrate were a β- d-anomeric 2-OH in the furanose ring, a 4-OH trans to the d-5-CH 2OH and a -CH 2OH substituent on C 2 ( trans to the 5-CH 2OH) which could be modified. The orientation of the hydroxyl on C-3 had only a limited effect.