Ketohexokinase in Microorganisms

在一个新辅基吡咯并喹啉醌生物合成的研究过程中，使用了许多甲基营养细菌，包括专性和兼性菌株，它们已知生长在D-果糖上，但不生长在D-葡萄糖上，以了解哪些类型的酶与吡咯并喹啉醌的生物合成有关。尽管己糖激酶和果糖激酶的酶活性很差，但有趣的是看到生物体可以在D-果糖上生长良好。对可能参与D-果糖代谢的酶进行了研究，发现大多数这类微生物的无细胞提取物中有较强的己酮糖激酶活性。通过涉及各种层析的方法从无细胞提取物中纯化酶。酶经纯化后得到单一蛋白条带，纯化倍数为1,000倍，得率为40%。即使当用稀释的酶溶液（例如每ml μ g蛋白质的水平）进行检查时，在70 ℃下加热酶溶液30分钟后也没有观察到明显的酶失活。通过以下证据，该酶催化D-果糖磷酸化产生D-果糖-1-磷酸的直接检测似乎是非常可能的。在反应混合物中没有检测到D-果糖-6-磷酸，因为包括磷酸己糖异构酶和D-葡萄糖-6-磷酸脱氢酶的偶联酶系统显示对反应产物没有反应，尽管由丙酮酸激酶和乳酸脱氢酶组成的偶联酶系统检测到ADP的生成。

In a course of studies on biosynthesis of a novel prosthetic group, pyrroloouinoline auinone, a number of methylotrophic bacteria, including obligate and facultatiie strains, which are known to grow on D-fructose but not on D-glucose, were used to see what kinds of enzymes are concerned in biosynthesis of pyrroloquinoline quinone. In spite of a poor enzyme activity of hexokinase and fructokinase, it is interesting to see that the organisms can grow well on D-fructose. After a survey ofenzymes possibly involved in D-fructose metabolism, a fairly strong enzyme activity of ketohexokinase was found in the cell free extract of most of such ibitroorganisms. The enzyme was purified from the cell free extract by a method involving various kinds of chromatography. The enzyme was finally purified to a single protein band with 1,000-fold and 40% yield. Even when examined with a diluted enzyme solution such a level of ug protein per ml, no appreciable inactivation of the enzyme was observed after 30 min heating the enzyme solution at 70ﾟC. A direct examination that the enzyme catalyzes phosphorylation of D-fructose yielding D-fructose-1-phosphate seemed to be quite probable by the following evidence. D-Fructose-6-phosphate was not detected in the reaction mixture because a coupling enzyme system including phosphohexose isomerase and D-glucose-6-phosphate dehydrogenase showed no response to the reaction product, though foreation of ADP was detected by a coupling enzyme system consisting of pyruvate kinase and lactate dehydrogenase.