Nitrilase catalyzes amide hydrolysis as well as nitrile hydrolysis

While amides were reported to be completely inert as substrates for all nit rilases reported to date, the nitrilase from Rhodococcus rhodochrous J1, wh ich catalyzes the hydrolytic cleavage of the C-N triple bond in nitrile to form acid and ammonium, was surprisingly found to catalyze hydrolysis of am ide to acid and ammonium stoichiometrically. This nitrilase exhibited a K-m of 2.94 mM for benzamide, similar to that for benzonitrile as the original substrate (2.10 mM), but the V-max for benzamide was six orders of magnitu de lower than that for benzonitrile. Benzamide inhibited the nitrilase reac tion in a reversible, apparently competitive manner. A mutant nitrilase con taining alanine or serine instead of Cys165, which is essential for nitrila se catalytic activity, showed no amidase activity. This observation demonst rated that Cys165 plays a crucial role in the hydrolysis of amides as well as nitriles. Together with some reports that certain nitrilases were previo usly noted to produce low amounts of amide as a by-product from nitrile, th e above unexpected findings suggested the existence of a common tetrahedral intermediate in the nitrilase reaction involving nitrile or amide as a sub strate. (C) 1998 Academic Press.