Nelp Micah T, Song Yang, Wysocki Vicki H, Bandarian Vahe
From the Department of Chemistry, University of Utah, Salt Lake City, Utah, 84112 and.
the Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210.
J Biol Chem. 2016 Apr 8;291(15):7822-9. doi: 10.1074/jbc.M115.704791. Epub 2016 Feb 10.
Nitrile hydratase metalloenzymes are unique and important biocatalysts that are used industrially to produce high value amides from their corresponding nitriles. After more than three decades since their discovery, the mechanism of this class of enzymes is becoming clear with evidence from multiple recent studies that the cysteine-derived sulfenato ligand of the active site metal serves as the nucleophile that initially attacks the nitrile. Herein we describe the first direct evidence from solution phase catalysis that the source of the product carboxamido oxygen is the protein. Using(18)O-labeled water under single turnover conditions and native high resolution protein mass spectrometry, we show that the incorporation of labeled oxygen into both product and protein is turnover-dependent and that only a single oxygen is exchanged into the protein even under multiple turnover conditions, lending significant support to proposals that the post-translationally modified sulfenato group serves as the nucleophile to initiate hydration of nitriles.
腈水合酶金属酶是一类独特且重要的生物催化剂，在工业上用于从相应的腈生产高价值的酰胺。自发现以来的三十多年里，这类酶的作用机制正逐渐明晰，多项近期研究表明，活性位点金属上由半胱氨酸衍生的亚磺酸盐配体作为亲核试剂，最初攻击腈。在此，我们描述了来自溶液相催化的首个直接证据，即产物羧酰胺氧的来源是蛋白质。在单周转条件下使用（18）O标记的水和天然高分辨率蛋白质质谱，我们表明标记氧掺入产物和蛋白质均依赖于周转，并且即使在多周转条件下，也只有一个氧交换到蛋白质中，这为翻译后修饰的亚磺酸盐基团作为亲核试剂引发腈水合反应的提议提供了有力支持。
