Takata H, Kuriki T, Okada S, Takesada Y, Iizuka M, Minamiura N, Imanaka T
Biochemical Research Laboratories, Ezaki Glico Co., Ltd., Osaka, Japan.
J Biol Chem. 1992 Sep 15;267(26):18447-52.
The transglycosylation reaction catalyzed by neopullulanase was analyzed. Radioactive oligosaccharides were produced when the enzyme acted on maltotriose in the presence of [U-14C]glucose. Some of the radioactive oligosaccharides had only alpha-(1----4)-glucosidic linkages, but others were suggested to have alpha-(1----6)-glucosidic linkages. The existence of alpha-(1----6)-glucosidic linkages in the products from maltotriose with neopullulanase was proven by proton NMR spectroscopy and methylation analysis. We previously reported that the one active center of neopullulanase catalyzes the hydrolysis of alpha-(1----4)- and alpha-(1----6)-glucosidic linkages (Kuriki, T., Takata, H., Okada, S., and Imanaka, T. (1991) J. Bacteriol. 173,6147-6152). These facts proved that neopullulanase catalyzed all four types of reactions: hydrolysis of alpha-(1----4)-glucosidic linkage, hydrolysis of alpha-(1----6)-glucosidic linkage, transglycosylation to form alpha-(1----4)-glucosidic linkage, and transglycosylation to form alpha-(1----6)-glucosidic linkage. The four reactions are typically catalyzed by alpha-amylase, pullulanase, cyclomaltodextrin glucanotransferase, and 1,4-alpha-D-glucan branching enzyme, respectively. These four enzymes have some structural similarities to one other, but reactions catalyzed by the enzymes are considered to be distinctive: the four reactions are individually catalyzed by each of the enzymes. The experimental results obtained from the analysis of the reaction of the neopullulanase exhibited that the four reactions can be catalyzed in the same mechanism.
对新普鲁兰酶催化的转糖基化反应进行了分析。当该酶在[U-14C]葡萄糖存在下作用于麦芽三糖时，产生了放射性寡糖。一些放射性寡糖仅具有α-(1→4)-糖苷键，但其他一些则被认为具有α-(1→6)-糖苷键。通过质子核磁共振光谱和甲基化分析证实了麦芽三糖与新普鲁兰酶反应产物中存在α-(1→6)-糖苷键。我们之前报道过，新普鲁兰酶的一个活性中心催化α-(1→4)-和α-(1→6)-糖苷键的水解（栗木哲也、高田浩、冈田诚、稻中稔（1991年）《细菌学杂志》173, 6147 - 6152）。这些事实证明新普鲁兰酶催化所有四种类型的反应：α-(1→4)-糖苷键的水解、α-(1→6)-糖苷键的水解、形成α-(1→4)-糖苷键的转糖基化反应以及形成α-(1→6)-糖苷键的转糖基化反应。这四种反应通常分别由α-淀粉酶、普鲁兰酶、环麦芽糊精葡糖基转移酶和1,4-α-D-葡聚糖分支酶催化。这四种酶彼此之间在结构上有一些相似之处，但它们催化的反应被认为是不同的：这四种反应分别由每种酶单独催化。对新普鲁兰酶反应分析获得的实验结果表明，这四种反应可以通过相同的机制进行催化。
