The roles of Glu186 and Glu380 in the catalytic reaction of soybean β-amylase
The roles of Glu186 and Glu380 in the catalytic reaction of soybean β-amylase
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DOI：
10.1016/j.jmb.2004.04.029
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发表时间：
2004-06-18
影响因子：
5.6
通讯作者：
Mikami, B
中科院分区：
文献类型：
--
作者：
Kang, YN;Adachi, M;Mikami, B
先前的研究表明，大豆β-淀粉酶的谷氨酸残基Glu186和Glu380分别作为总酸和总碱催化剂起着关键作用。为了确定Glu186和Glu380的作用，将每个残基突变为谷氨酰胺残基，并分别在1.6埃和1.9埃的分辨率下测定底物(E186Q/麦芽五糖)和产物(E380Q/麦芽糖)的晶体结构。与野生型酶相比，两种突变酶的活性分别降低了16,000倍和37,000倍。E186Q/麦芽糖复合体的晶体结构显示在-2到+3亚基上有一个明确的五糖单元。在E380Q/麦芽糖复合体中，两个麦芽糖分子结合在亚基-2到-1和+2到+3上，而在野生型/麦芽糖复合体中，它们依次结合到-2到-1和+1到+2亚基上。在E380Q/麦芽糖复合体中，葡萄糖残基-1的构象为稳定的C-4(1)α-异构体，而在野生型/麦芽糖复合体中观察到扭曲的环状构象。Gln380的侧链移动到野生型酶中可能攻击水分子的位置，导致E380Q突变体失活，麦芽糖分子的结合模式发生变化。这些结果证实了Glu186在给予质子给底物的糖苷氧中所起的关键作用，以及Glu380在激活攻击性水分子中所起的关键作用。E186Q/麦芽五糖和E380Q/麦芽糖的骨架在Thr342方面的差异表明，Thr342的侧链可能在糖苷键断裂后稳定了去质子型的Glu186。(C)2004爱思唯尔有限公司。保留所有权利。
It has previously been suggested that the glutamic acid residues Glu186 and Glu380 of soybean beta-amylase play critical roles as a general acid and a general base catalyst, respectively. In order to confirm the roles of Glu186 and Glu380, each residue was mutated to a glutamine residue and the crystal structures of the substrate (E186Q/maltopentaose) and product (E380Q/maltose) complexes were determined at resolutions of 1.6 Angstrom and 1.9 Angstrom, respectively. Both mutant enzymes exhibited 16,000- and 37,000-fold decreased activity relative to that of the wild-type enzyme. The crystal structure of the E186Q/maltopentaose complex revealed an unambiguous five-glucose unit at subsites -2 to +3. Two maltose molecules bind on subsites -2 to -1 and +2 to +3 in the E380Q/maltose complex, whereas they bind in tandem to -2 to -1 and +1 to +2 in the wild-type/maltose complex. The conformation of the glucose residue at subsite-1 was identified as a stable C-4(1) alpha-anomer in the E380Q/maltose complex, whereas a distorted ring conformation was observed in the wild-type/maltose complex. The side-chain movement of Gln380 to the position of a putative attacking water molecule seen in the wild-type enzyme caused the inactivation of the E380Q mutant and an altered binding pattern of maltose molecules. These results confirm the critical roles played by Glu186 in the donation of a proton to the glycosidic oxygen of the substrate, and by Glu380 in the activation of an attacking water molecule. The observed difference between the backbones of E186Q/maltopentaose and E380Q/maltose in terms of Thr342 suggests that the side-chain of Thr342 may stabilize the deprotonated form of Glu186 after the cleavage of the glycosidic bond. (C) 2004 Elsevier Ltd. All rights reserved.