Nishimasu Hiroshi, Fushinobu Shinya, Shoun Hirofumi, Wakagi Takayoshi
Department of Biotechnology, the University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Department of Biotechnology, the University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
J Biol Chem. 2007 Mar 30;282(13):9923-9931. doi: 10.1074/jbc.M610678200. Epub 2007 Jan 17.
Hexokinase catalyzes the phosphorylation of glucose to glucose 6-phosphate by using ATP as a phosphoryl donor. Recently, we identified and characterized an ATP-dependent hexokinase (StHK) from the hyperthermophilic archaeon Sulfolobus tokodaii, which can phosphorylate a broad range of sugar substrates, including glucose, mannose, glucosamine, and N-acetylglucosamine. Here we present the crystal structures of StHK in four different forms: (i) apo-form, (ii) binary complex with glucose, (iii) binary complex with ADP, and (iv) quaternary complex with xylose, Mg(2+), and ADP. Forms i and iii are in the open state, and forms ii and iv are in the closed state, indicating that sugar binding induces a large conformational change, whereas ADP binding does not. The four different crystal structures of the same enzyme provide "snapshots" of the conformational changes during the catalytic cycle. StHK exhibits a core fold characteristic of the hexokinase family, but the structures of several loop regions responsible for substrate binding are significantly different from those of other known hexokinase family members. Structural comparison of StHK with human N-acetylglucosamine kinase and other hexokinases provides an explanation for the ability of StHK to phosphorylate both glucose and N-acetylglucosamine. A Mg(2+) ion and coordinating water molecules are well defined in the electron density of the quaternary complex structure. This structure represents the first direct visualization of the binding mode for magnesium to hexokinase and thus allows for a better understanding of the catalytic mechanism proposed for the entire hexokinase family.
己糖激酶以ATP作为磷酰基供体，催化葡萄糖磷酸化为6-磷酸葡萄糖。最近，我们从嗜热古菌硫磺矿硫化叶菌中鉴定并表征了一种ATP依赖性己糖激酶（StHK），它可以磷酸化多种糖类底物，包括葡萄糖、甘露糖、葡糖胺和N-乙酰葡糖胺。在此，我们展示了StHK四种不同形式的晶体结构：（i）无配体形式，（ii）与葡萄糖的二元复合物，（iii）与ADP的二元复合物，以及（iv）与木糖、Mg(2+)和ADP的四元复合物。形式i和iii处于开放状态，而形式ii和iv处于关闭状态，这表明糖类结合会诱导较大的构象变化，而ADP结合则不会。同一酶的四种不同晶体结构提供了催化循环过程中构象变化的“快照”。StHK展现出己糖激酶家族的核心折叠特征，但负责底物结合的几个环区结构与其他已知己糖激酶家族成员的结构有显著差异。将StHK与人类N-乙酰葡糖胺激酶及其他己糖激酶进行结构比较，为StHK能够磷酸化葡萄糖和N-乙酰葡糖胺的能力提供了解释。在四元复合物结构的电子密度图中，Mg(2+)离子和配位水分子清晰可辨。该结构首次直接展示了镁与己糖激酶的结合模式，从而有助于更好地理解整个己糖激酶家族的催化机制。
