QUATERNARY LIGAND-BINDING TO AROMATIC RESIDUES IN THE ACTIVE-SITE GORGE OF ACETYLCHOLINESTERASE
QUATERNARY LIGAND-BINDING TO AROMATIC RESIDUES IN THE ACTIVE-SITE GORGE OF ACETYLCHOLINESTERASE
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DOI：
10.1073/pnas.90.19.9031
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发表时间：
1993-10-01
影响因子：
11.1
通讯作者：
SUSSMAN, JL
中科院分区：
文献类型：
--
作者：
HAREL, M;SCHALK, I;SUSSMAN, JL
利用x射线晶体学和光亲和标记技术研究了鱼雷乙酰胆碱酯酶（EC 3.1.1.7）与季配体的结合位点。在2.8埃分辨率下测定了配体配合物的晶体结构。在与邻苯二酚的配合物中，配体的季氮与Trp-84的吲哚相互作用，其间羟基与催化三联体的两个成员Ser-200和His-440显示分叉氢键。在与塔克林的络合物中，吖啶与Trp- 84的吲哚叠在一起。双季配体十甲基铵沿通向活性位点的狭窄峡谷取向；在峡谷顶端附近，一个与Trp-84的吲哚相对，另一个与Trp-279的吲哚相对。这三种配合物之间唯一的主要构象区别在于ph -330的苯基环的取向。在十甲铵复合体中，它与峡谷的表面平行；在另外两种配合物中，它被定位于与结合的配体接触。这种密切的相互作用通过光敏探针h -3标记的p-（N，N-二甲氨基）苯二氮唑氟硼酸盐的光亲和标记得到证实，该探针在活性位点内主要标记了ph -330。还观察了Trp-279的标记。每摩尔AcChoEase失活1摩尔标签，表明Trp-279的标记和ph -330的标记是相互排斥的。结构和化学数据共同表明芳香基团作为四元配体结合位点的重要作用，它们提供了补充证据，证明Trp-84和ph -330位于活性位点的“阴离子”亚位，而Trp-279位于“外周”阴离子位点。
Binding sites of Torpedo acetylcholinesterase (EC 3.1.1.7) for quaternary ligands were investigated by x-ray crystallography and photoaffinity labeling. Crystal structures of complexes with ligands were determined at 2.8-angstrom resolution. In a complex with edrophonium, the quaternary nitrogen of the ligand interacts with the indole of Trp-84, and its m-hydroxyl displays bifurcated hydrogen bonding to two members of the catalytic triad, Ser-200 and His-440. In a complex with tacrine, the acridine is stacked against the indole of Trp- 84. The bisquaternary ligand decamethonium is oriented along the narrow gorge leading to the active site; one quaternary group is apposed to the indole of Trp-84 and the other to that of Trp-279, near the top of the gorge. The only major conformational difference between the three complexes is in the orientation of the phenyl ring of Phe-330. In the decamethonium complex it lies parallel to the surface of the gorge; in the other two complexes it is positioned to make contact with the bound ligand. This close interaction was confirmed by photoaffinity labeling by the photosensitive probe H-3-labeled p-(N,N-dimethylamino)benzenediazonium fluoroborate, which labeled, predominantly, Phe-330 within the active site. Labeling of Trp-279 was also observed. One mole of label is incorporated per mole of AcChoEase inactivated, indicating that labeling of Trp-279 and that of Phe-330 are mutually exclusive. The structural and chemical data, together, show the important role of aromatic groups as binding sites for quaternary ligands, and they provide complementary evidence assigning Trp-84 and Phe-330 to the ''anionic'' subsite of the active site and Trp-279 to the ''peripheral'' anionic site.