Purification and Properties of Hamamelosekinase

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Abstract
Hamamelose kinase (ATP:hamamelose 21-phosphotransferase) was purified from a crude extract of Kluyvera citrophila 627 (Enterobacteriaceae), which was grown on D-hamamelose. Ammonium sulfate fractionation and 2-fold chromatography on DEAE-cellulose resulted in a 51-fold purification of the enzyme. Neither glucose kinase nor significant ATPase activity could be detected in the pure preparation. Besides D-hamamelose only D-hamamelitol was utized as a substrate. The latter was phosphorylated at a very low rate. The MW of the enzyme as estimated by gel chromatography is 21,000. The Km values for hamamelose and ATP were 3 mM and 2.5 mM, respectively. The pH optimum was 7.5. In contrast to hexokinase, purified hamamelose kinase is very labile and could only be stabilized by addition of its substrate D-hamamelose. The most unusual property compared to yeast hexokinase is a pronounced substrate inhibition by hamamelose (> 5 mM) and ATP (> 7 mM), respectively, which could be due to economic utilization of the nutrient. Hamamelose kinase and glucose kinase are inducible by growing the microorganisms on the corresponding monosaccharides.

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