Nitrilase catalyzes amide hydrolysis as well as nitrile hydrolysis.

While amides were reported to be completely inert as substrates for all nitrilases reported to date, the nitrilase from Rhodococcus rhodochrous J1, which catalyzes the hydrolytic cleavage of the C-N triple bond in nitrile to form acid and ammonium, was surprisingly found to catalyze hydrolysis of amide to acid and ammonium stoichiometrically. This nitrilase exhibited a Km of 2.94 mM for benzamide, similar to that for benzonitrile as the original substrate (2.10 mM), but the Vmax for benzamide was six orders of magnitude lower than that for benzonitrile. Benzamide inhibited the nitrilase reaction in a reversible, apparently competitive manner. A mutant nitrilase containing alanine or serine instead of Cys165, which is essential for nitrilase catalytic activity, showed no amidase activity. This observation demonstrated that Cys165 plays a crucial role in the hydrolysis of amides as well as nitriles. Together with some reports that certain nitrilases were previously noted to produce low amounts of amide as a by-product from nitrile, the above unexpected findings suggested the existence of a common tetrahedral intermediate in the nitrilase reaction involving nitrile or amide as a substrate.