National Center for
3WVD: Crystal Structure Of Nitrile Hydratase Mutant Br56k Complexed With Trimethylacetonitrile, Photo-activated For 50 Min
Time-Resolved Crystallography of the Reaction Intermediate of Nitrile Hydratase: Revealing a Role for the Cysteinesulfenic Acid Ligand as a Catalytic Nucleophile
Angew. Chem. Int. Ed. Engl. (2015) 54 p.10763-10767
The reaction mechanism of nitrile hydratase (NHase) was investigated using time-resolved crystallography of the mutant NHase, in which betaArg56, 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 A). In combination with FTIR analyses of NHase following hydration in H2 (18) O, we propose that the metal-coordinated substrate is nucleophilically attacked by the O(SO(-) ) atom of alphaCys114-SO(-) , followed by nucleophilic attack of the S(SO(-) ) atom by a betaArg56-activated water molecule to release the product amide and regenerate alphaCys114-SO(-) .