Catalytic Mechanism of Nitrile Hydratase Proposed by Time-resolved X-ray Crystallography Using a Novel Substrate, tert-Butylisonitrile
Catalytic Mechanism of Nitrile Hydratase Proposed by Time-resolved X-ray Crystallography Using a Novel Substrate, tert-Butylisonitrile
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DOI：
10.1074/jbc.m806577200
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发表时间：
2008-12-26
影响因子：
4.8
通讯作者：
Odaka, Masafumi
中科院分区：
文献类型：
--
作者：
Hashimoto, Koichi;Suzuki, Hiroyuki;Odaka, Masafumi
氰基水合酶(NHase)有一个特殊的铁或钴催化中心，它有两个被氧化的半胱氨酸配体，半胱氨酸亚磺酸和半胱氨酸-磺酸，催化氰类化合物水合生成酰胺。最近，我们发现红球红球菌N771的氨基转移酶具有额外的催化活性，可以将叔丁基异腈(TBuNC)转化为叔丁胺。利用tBuNC的缓慢反应性和硝酸氨酶的光反应性，我们首次用时间分辨x射线结晶学对NHase的催化机理提供了结构证据。通过衰减全反射傅里叶变换红外光谱监测反应，异腈碳生成的产物为CO分子。硝化失活氨酶晶体用tBuNC浸泡。催化反应由光诱导脱氮引发，由闪冷终止。TBuNC首先被捕获在铁中心上方的疏水口袋中，然后在120min与铁离子配位。在440min时，tBuNC的电子密度发生了显著变化，并在α-半胱氨酸(114)的异腈碳和亚硫酸盐氧附近观察到一个新的电子密度。这些结果表明，底物是与铁配位的，然后被α-半胱氨酸(114)-SOH活化的溶剂分子攻击。
Nitrile hydratases (NHases) have an unusual iron or cobalt catalytic center with two oxidized cysteine ligands, cysteinesulfinic acid and cysteine-sulfenic acid, catalyzing the hydration of nitriles to amides. Recently, we found that the NHase of Rhodococcus erythropolis N771 exhibited an additional catalytic activity, converting tert-butylisonitrile (tBuNC) to tert-butylamine. Taking advantage of the slow reactivity of tBuNC and the photoreactivity of nitrosylated NHase, we present the first structural evidence for the catalytic mechanism of NHase with time-resolved x-ray crystallography. By monitoring the reaction with attenuated total reflectance Fourier transform infrared spectroscopy, the product from the isonitrile carbon was identified as a CO molecule. Crystals of nitrosylated inactive NHase were soaked with tBuNC. The catalytic reaction was initiated by photo-induced denitrosylation and stopped by flash cooling. tBuNC was first trapped at the hydrophobic pocket above the iron center and then coordinated to the iron ion at 120 min. At 440 min, the electron density of tBuNC was significantly altered, and a new electron density was observed near the isonitrile carbon as well as the sulfenate oxygen of alpha Cys(114). These results demonstrate that the substrate was coordinated to the iron and then attacked by a solvent molecule activated by alpha Cys(114)-SOH.