Purification and properties of hamamelosekinase.
Hamamelosekinase (ATP:hamamelose 2(1)-phosphotransferase) was purified from a crude extract of Kluyvera citrophila 627 (Enterobacteriaeceae) which has been grown on D-hamamelose. Ammonium-sulfate fractionation and twofold chromatography on DEAE-cellulose resulted in a 51-fold purification of the enzyme. Neither glucosekinase nor significant ATPase activity could be detected in the pure preparation. Besides D-hamamelose only D-hamamelitol was utilized as a substrate; however, the latter was phosphorylated at a very low rate. The molecular weight of the enzyme as estimated by gel chromatography is 21 000. The Km values for hamamelose and ATP were 3 mM nd 2.5 mM, respectively. The pH optimum was found at 7.5. In contrast to hexokinase, purified hamamelosekinase is very labile and could only be stabilized by addition of its substrate D-hamamelose. The most unusual property with respect to yeast hexokinase is a pronounced substrate inhibiton by hamamelose (> 5mM) and ATP (> 7mM), respectively, which could be interpreted as due to an economic utilization of the nutrient. Hamamelosekinase as well as glucosekinase are inducible by growing the microorganisms on the corresponding monosaccharides.
D002848:Chromatography, DEAE-Cellulose; D004755:Enterobacteriaceae / Q000201:enzymology; D007700:Kinetics; D008970:Molecular Weight; D010770:Phosphotransferases / Q000302:isolation & purification / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor); D013379:Substrate Specificity
D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C026271:hamamelose kinase