Aspects of antibody-catalyzed primary amide hydrolysis.

Because there are many known C-terminally amidated peptides of biological importance, there is great potential in medicine and organic synthesis for antibodies that catalyze primary amide bond hydrolysis or formation. We characterized a catalytic antibody, 13D11, raised to a phosphinate hapten, that hydrolyzed the primary amide of a dansyl-alkylated derivative of (R)-phenylalaninamide (DNS-(R)F-NH2). At pH 9.0, 13D11 hydrolyzed DNS-(R)F-NH2 with a kcat of 1.65 x 10(-7) s-1 (kcat/kuncat = 132) and a Km of 432 microM, and was stereospecifically hapten-inhibited (Ki = 14.0 microM). Control experiments indicated that the catalytic activity was not the result of a contaminating protease. In accordance with the hapten being a transition-state analog of base hydrolysis, the rate of DNS-(R)F-NH2 hydrolysis increased with hydroxide concentration to an optimum pH of 9.5. Above pH 9.5, activity declined rapidly suggesting the antibody was inactivated during the long incubation period. This work demonstrates the feasibility of generating catalytic antibodies to hydrolyze unactivated amide bonds without cofactor assistance.