Separation and Characterization of Four Hexose Kinases from Developing Maize Kernels

Abstract

Four forms of hexose kinase activity from developing maize (Zea mays L.) kernels have been separated by ammonium sulfate precipitation, gel filtration chromatography, blue-agarose chromatography, and ion exchange chromatography. Two of these hexose kinases utilized d-glucose most effectively and are classified as glucokinases (EC 2.7.1.2). The other two hexose kinases utilized only d-fructose and are classified as fructokinases (EC 2.7.1.4). All hexose kinases analyzed had broad pH optima between 7.5 and 9.5 with optimal activity at pH 8.5. The two glucokinases differed in substrate affinities. One form had low K(m) values [K(m)(glucose) = 117 micromolar, K(m)(ATP) = 66 micromolar] whereas the other form had much higher K(m) values [K(m)(glucose) = 750 micromolar, K(m)(ATP) = 182 micromolar]. Both fructokinases had similar substrate saturation responses. The K(m)(fructose) was about 130 micromolar and the K(m)(ATP) was about 700 micromolar. Both exhibited uncompetitive substrate inhibition by fructose [K(i)(fructose) = 1.40 to 2.00 millimolar]. ADP inhibited all four hexose kinase activities, whereas sugar phosphates had little effect on their activities. The data suggest that substrate concentrations are an important factor controlling hexose kinase activity in situ.