Time-resolved crystal structures of the reaction intermediate of nitrile hydrase reveal a role for the cystein-sulfenic acid ligand as a catalyst nucleophile
Time-resolved crystal structures of the reaction intermediate of nitrile hydrase reveal a role for the cystein-sulfenic acid ligand as a catalyst nucleophile
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腈水合酶反应中间体的时间分辨晶体结构揭示了半胱氨酸-磺酸配体作为催化剂亲核试剂的作用
DOI：
10.1002/anie.201502731
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
and Masafumi Odaka
中科院分区：
文献类型：
--
作者：
Yasuaki Yamanaka;Yuki Kato;Koichi Hashimoto;Keisuke Iida;Kazuo Nagasawa;Hiroshi Nakayama;Naoshi Dohmae;Keiichi Noguchi;Takumi Noguchi;Masafumi Yohda;and Masafumi Odaka
利用时间分辨晶体技术研究了腈水合酶（NHase）突变体的反应机理。突变体中，与两个翻译后氧化的半胱氨酸配体有氢键的βArg56被赖氨酸取代，戊腈为底物。在高分辨率下测定了反应中间体的晶体结构（1.2-1.3 Å）。结合H218O水化后NHase的FTIR分析，我们提出金属配位底物被αCys114‐SO−的O（SO−）原子亲核攻击，然后被βArg56活化的水分子亲核攻击S（SO−）原子释放产物酰胺并再生αCys114‐SO−。
The reaction mechanism of nitrile hydratase (NHase) was investigated using time‐resolved crystallography of the mutant NHase, in which βArg56, strictly conserved and hydrogen bonded to the two post‐translationally oxidized cysteine ligands, was replaced by lysine, and pivalonitrile was the substrate. The crystal structures of the reaction intermediates were determined at high resolution (1.2–1.3 Å). In combination with FTIR analyses of NHase following hydration in H218O, we propose that the metal‐coordinated substrate is nucleophilically attacked by the O(SO−) atom of αCys114‐SO−, followed by nucleophilic attack of the S(SO−) atom by a βArg56‐activated water molecule to release the product amide and regenerate αCys114‐SO−.