title: A Protein-derived Oxygen Is the Source of the Amide Oxygen of Nitrile Hydratases.
abstract: 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.
authors: From the Department of Chemistry, University of Utah, Salt Lake City, Utah, 84112 and.
authors: Micah T
authors: MT
authors: Nelp
authors: the Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210.
authors: Yang
authors: Y
authors: Song
authors: the Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210.
authors: Vicki H
authors: VH
authors: Wysocki
authors: From the Department of Chemistry, University of Utah, Salt Lake City, Utah, 84112 and vahe@chem.utah.edu.
authors: Vahe
authors: V
authors: Bandarian
mesh_terms: D000192:Actinomycetales / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D006836:Hydro-Lyases / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D010100:Oxygen / Q000032:analysis / Q000378:metabolism*; D011741:Pyrimidine Nucleosides / Q000378:metabolism; D014127:Toyocamycin / Q000378:metabolism
chemical_list: D011741:Pyrimidine Nucleosides; C005699:sangivamycin; D006836:Hydro-Lyases; C048831:nitrile hydratase; D014127:Toyocamycin; D010100:Oxygen
keywords: enzyme; enzyme catalysis; enzyme mechanism; metalloenzyme; nitrile hydratase; post-translational modification (PTM); protein chemical modification