Spectroscopic and Computational Studies of Nitrile Hydratase: Insights into Geometric and Electronic Structure and the Mechanism of Amide Synthesis.
Spectroscopic and Computational Studies of Nitrile Hydratase: Insights into Geometric and Electronic Structure and the Mechanism of Amide Synthesis.
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DOI：
10.1039/c5sc02012c
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发表时间：
2015-11-01
期刊：
影响因子：
8.4
通讯作者：
Solomon EI
中科院分区：
文献类型：
--
作者：
Light KM;Yamanaka Y;Odaka M;Solomon EI
除了活化配位的腈之外，腈水合酶还利用配位的次磺酸盐配体作为良好定向的亲核试剂来形成五元中间体，该五元中间体随后经受H2O的攻击以最终形成酰胺产物。腈水合酶（Nitrile hydratases，NHases）是催化腈水合为酰胺的单核非血红素酶。NHase是不寻常的，因为它利用低自旋（LS）FeIII中心和独特的配体组，该配体组由两个去质子化的骨架酰胺、基于半胱氨酸的次磺酸（RSO（H））和亚磺酸（RSO 2-）以及与外源配体位点反式的未修饰的半胱氨酸组成。电子顺磁共振（EPR），磁圆二色性（MCD）和低温吸收（LT-Abs）光谱用于确定丁酸结合（NHaseBA）和活性（NHaseAq）NHase的几何和电子结构。这些数据校准DFT模型，然后扩展到探讨腈水合的机制，由NHase。特别地，腈通过与LS FeIII配位而活化，并且发现亚磺酸酯基团被去质子化，并且是比水明显更好的亲核试剂，其可以攻击配位腈以形成环状物质。攻击的次磺酸酯S原子的环状物种是有利的，并导致一个较低的动力学势垒比攻击水的协调，未环化的腈，而攻击的环状物种的C是不利的。独特的配体集和低自旋性质的腈水合酶活性位点的功能的作用也进行了探讨。研究发现，氧化的硫醇配体是维持LS态的关键，这在腈基的结合和活化中是重要的。骨架酰胺化物配体的主要作用似乎是作为螯合物，保持亚磺酸盐正确定向，用于对配位底物的亲核攻击。
In addition to its activation of coordinated nitriles, nitrile hydratase utilizes a coordinated sulfenate ligand as a well-oriented nucleophile to form a five-membered intermediate which subsequently undergoes attack by H2O to ultimately form the amide product. Nitrile hydratases (NHases) are mononuclear nonheme enzymes that catalyze the hydration of nitriles to amides. NHase is unusual in that it utilizes a low-spin (LS) FeIII center and a unique ligand set comprised of two deprotonated backbone amides, cysteine-based sulfenic acid (RSO(H)) and sulfinic acid (RSO2–), and an unmodified cysteine trans to an exogenous ligand site. Electron paramagnetic resonance (EPR), magnetic circular dichroism (MCD) and low-temperature absorption (LT-Abs) spectroscopies are used to determine the geometric and electronic structures of butyrate-bound (NHaseBA) and active (NHaseAq) NHase. These data calibrate DFT models, which are then extended to explore the mechanism of nitrile hydration by NHase. In particular, the nitrile is activated by coordination to the LS FeIII and the sulfenate group is found to be deprotonated and a significantly better nucleophile than water that can attack the coordinated nitrile to form a cyclic species. Attack at the sulfenate S atom of the cyclic species is favorable and leads to a lower kinetic barrier than attack by water on coordinated, uncyclized nitrile, while attack at the C of the cyclic species is unfavorable. The roles of the unique ligand set and low-spin nature of the NHase active site in function are also explored. It is found that the oxidized thiolate ligands are crucial to maintaining the LS state, which is important in the binding and activation of nitrile susbtrates. The dominant role of the backbone amidate ligands appears to be as a chelate in keeping the sulfenate properly oriented for nucleophilic attack on the coordinated substrate.