THE CATALYTIC MECHANISM OF AMIDASE ALSO INVOLVES NITRILE HYDROLYSIS

The amidase from Rhodococcus rhonochrous J1, which hydrolyzes an amide to an acid and ammonium, mas surprisingly found to catalyze the hydro lytic cleavage of the C-N triple bond in a nitrile to form an acid and ammonium stoichiometrically. The amidase exhibited a K-m of 3.26 mM f or benzonitrile in contrast to that of 0.15 mM for benzamide as the or iginal substrate, but the V-max for benzonitrile was about 1/6000 of t hat for benzamide. A mutant amidase containing alanine instead of Ser( 195), which is essential for amidase catalytic activity showed no nitr ilase activity, demonstrating that this residue plays a crucial role i n the hydrolysis of nitriles as well as amides. (C) 1998 Federation of European Biochemical Societies.