Substrate and product trafficking through the active center gorge of acetylcholinesterase analyzed by crystallography and equilibrium binding
Substrate and product trafficking through the active center gorge of acetylcholinesterase analyzed by crystallography and equilibrium binding
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DOI：
10.1074/jbc.m603018200
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发表时间：
2006-09-29
影响因子：
4.8
通讯作者：
Marchot, Pascale
中科院分区：
文献类型：
--
作者：
Bourne, Yves;Radic, Zoran;Marchot, Pascale
乙酰胆碱酯酶（AChE）是自然界中最有效的酶之一，它催化乙酰胆碱水解的反应发生在一个深而窄的活性中心峡谷的底部。在入口处的峡谷，周边的阴离子网站提供了一个结合位点的变构配体，包括基板。迄今为止，没有结构信息基板进入活性中心从周边网站的乙酰胆碱酯酶或其随后的出口已被报道。小鼠AChE和具有取代的催化丝氨酸（S203 A）的失活小鼠AChE突变体在与四种底物的各种复合物中的互补晶体结构（乙酰胆碱、乙酰硫代胆碱、琥珀酰二胆碱和丁酰硫代胆碱），两种不可水解的底物类似物（间-（N，N，N-三甲基铵基）三氟苯乙酮和4-酮戊基三甲基铵），和一种反应产物（胆碱）在2.05-2.65埃分辨率范围内溶解。这些结构，支持结合和抑制数据上获得的相同的复合物，揭示了连续的位置和方向的基板绑定到外围网站，并进行峡谷内的活性位点，构象的假定过渡态酰化和酰基酶中间体，和位置和方向的解离和外出的产品。此外，乙酰胆碱酯酶突变体的结构与乙酰硫胆碱和琥珀酰胆碱复合物揭示了额外的底物结合位点的酶表面上，远端的峡谷入口。因此，我们提供了一套全面的结构快照的步骤，导致催化的中间体和潜在的调节底物结合到酶表面的各种变构位点。
Hydrolysis of acetylcholine catalyzed by acetylcholinesterase (AChE), one of the most efficient enzymes in nature, occurs at the base of a deep and narrow active center gorge. At the entrance of the gorge, the peripheral anionic site provides a binding locus for allosteric ligands, including substrates. To date, no structural information on substrate entry to the active center from the peripheral site of AChE or its subsequent egress has been reported. Complementary crystal structures of mouse AChE and an inactive mouse AChE mutant with a substituted catalytic serine (S203A), in various complexes with four substrates (acetylcholine, acetylthiocholine, succinyldicholine, and butyrylthiocholine), two non-hydrolyzable substrate analogues (m-(N, N, N-trimethylammonio)trifluoroacetophenone and 4-ketoamyltrimethylammonium), and one reaction product (choline) were solved in the 2.05-2.65-angstrom resolution range. These structures, supported by binding and inhibition data obtained on the same complexes, reveal the successive positions and orientations of the substrates bound to the peripheral site and proceeding within the gorge toward the active site, the conformations of the presumed transition state for acylation and the acyl-enzyme intermediate, and the positions and orientations of the dissociating and egressing products. Moreover, the structures of the AChE mutant in complexes with acetylthiocholine and succinyldicholine reveal additional substrate binding sites on the enzyme surface, distal to the gorge entry. Hence, we provide a comprehensive set of structural snapshots of the steps leading to the intermediates of catalysis and the potential regulation by substrate binding to various allosteric sites at the enzyme surface.