Catalytic Mechanism of Nitrile Hydratase Subsequent to Cyclic Intermediate Formation: A QM/MM Study

The catalytic mechanism of an Fe-containing nitrile hydratase (NHase) subsequent to the formation of a cyclic intermediate was investigated using a hybrid quantum mechanics/molecular mechanics (QM/MM) method. We identified the following mechanism: (i) proton transfer from beta Tyr72 to the substrate via alpha Ser113, and cleavage of the S-O bond of alpha Cys114-SO- and formation of a disulfide bond between alpha Cys109 and alpha Cys114; (ii) direct attack of a water molecule on the sulfur atom of alpha Cys114, which resulted in the generation of both an imidic acid and a renewed sulfenic cysteine; and (iii) isomerization of the imidic acid to the amide. In addition, to clarify the role of beta Arg56K, which is one of the essential amino residues in the enzyme, we analyzed a beta R56K mutant in which beta Arg56 was replaced by Lys. The results suggest that beta Arg56 is necessary for the formation of disulfide intermediate by stabilizing the cleavage of the S-O bond via a hydrogen bond with the oxygen atom of alpha Cys114-SO-.