The active site sulfenic acid ligand in nitrile hydratases can function as a nucleophile.
The active site sulfenic acid ligand in nitrile hydratases can function as a nucleophile.
复制标题
DOI：
10.1021/ja410462j
复制
发表时间：
2014-01-29
影响因子：
15
通讯作者：
Holz R
中科院分区：
文献类型：
--
作者：
Martinez S;Wu R;Sanishvili R;Liu D;Holz R
腈水合酶 (NHase) 在环境温度和生理 pH 值下催化腈水合成相应的具有商业价值的酰胺。 NHase 酶的几种反应机制已被提出；然而，亲核试剂的来源仍然是个谜。硼酸已被证明是许多水解酶的有效抑制剂，因为硼的开放壳使其能够从三角平面 (sp2) 形式扩展到四面体形式 (sp3)。因此，我们通过动力学和 X 射线晶体学研究了硼酸对嗜热假诺卡氏菌 JCM 3095 (PtNHase) 的 Co 型 NHase 的抑制作用。 1-丁硼酸 (BuBA) 和苯基硼酸 (PBA) 都是 PtNHase 的有效竞争性抑制剂。与 PtNHase 复合的 BuBA 和 PBA 的 X 射线晶体结构以 1.5、1.6 和 1.2 Å 分辨率进行解析和细化。所得的 PtNHase-硼酸复合物代表了反应中间体的“快照”，并暗示半胱氨酸-次磺酸配体作为催化亲核试剂，这是 αCys113-OH 次磺酸配体迄今为止未知的作用。基于这些数据，提出了 NHase 使腈水合的新作用机制。
Nitrile hydratase (NHase) catalyzes the hydration of nitriles to their corresponding commercially valuable amides at ambient temperatures and physiological pH. Several reaction mechanisms have been proposed for NHase enzymes; however, the source of the nucleophile remains a mystery. Boronic acids have been shown to be potent inhibitors of numerous hydrolytic enzymes due to the open shell of boron, which allows it to expand from a trigonal planar (sp2) form to a tetrahedral form (sp3). Therefore, we examined the inhibition of the Co-type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase) by boronic acids via kinetics and X-ray crystallography. Both 1-butaneboronic acid (BuBA) and phenylboronic acid (PBA) function as potent competitive inhibitors of PtNHase. X-ray crystal structures for BuBA and PBA complexed to PtNHase were solved and refined at 1.5, 1.6, and 1.2 Å resolution. The resulting PtNHase–boronic acid complexes represent a “snapshot” of reaction intermediates and implicate the cysteine-sulfenic acid ligand as the catalytic nucleophile, a heretofore unknown role for the αCys113–OH sulfenic acid ligand. Based on these data, a new mechanism of action for the hydration of nitriles by NHase is presented.
登录
查看更多内容
影响因子：
15
作者：
Arakawa, Takatoshi;Kawano, Yoshiaki;Odaka, Masafumi
通讯作者：
Odaka, Masafumi
影响因子：
15
作者：
Dey, A;Chow, M;Solomon, EI
通讯作者：
Solomon, EI
影响因子：
4.8
作者：
Gumataotao, Natalie;Kuhn, Misty L.;Holz, Richard C.
通讯作者：
Holz, Richard C.
影响因子：
5
作者：
Petrillo, KL;Wu, SJ;Payne, MS
通讯作者：
Payne, MS
影响因子：
2.9
作者：
De Paola, CC;Bennett, B;Petsko, GA
通讯作者：
Petsko, GA