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Time‐Resolved Crystallography of the Reaction Intermediate of Nitrile Hydratase: Revealing a Role for the Cysteinesulfenic Acid Ligand as a Catalytic Nucleophile
Author(s) -
Yamanaka Yasuaki,
Kato Yuki,
Hashimoto Koichi,
Iida Keisuke,
Nagasawa Kazuo,
Nakayama Hiroshi,
Dohmae Naoshi,
Noguchi Keiichi,
Noguchi Takumi,
Yohda Masafumi,
Odaka Masafumi
Publication year - 2015
Publication title -
angewandte chemie international edition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.831
H-Index - 550
eISSN - 1521-3773
pISSN - 1433-7851
DOI - 10.1002/anie.201502731
Subject(s) - nitrile hydratase , chemistry , nucleophile , nitrile , ligand (biochemistry) , substrate (aquarium) , catalysis , molecule , hydrate , amide , stereochemistry , crystal structure , lyase , crystallography , enzyme , organic chemistry , biochemistry , biology , receptor , ecology
The reaction mechanism of nitrile hydratase (NHase) was investigated using time‐resolved crystallography of the mutant NHase, in which βArg56, strictly conserved and hydrogen bonded to the two post‐translationally oxidized cysteine ligands, was replaced by lysine, and pivalonitrile was the substrate. The crystal structures of the reaction intermediates were determined at high resolution (1.2–1.3 Å). In combination with FTIR analyses of NHase following hydration in H 2 18 O, we propose that the metal‐coordinated substrate is nucleophilically attacked by the O(SO − ) atom of αCys114‐SO − , followed by nucleophilic attack of the S(SO − ) atom by a βArg56‐activated water molecule to release the product amide and regenerate αCys114‐SO − .