ASPECTS OF ANTIBODY-CATALYZED PRIMARY AMIDE HYDROLYSIS

Because there are many known C-terminally amidated peptides of biologi cal importance, there is great potential in medicine and organic synth esis for antibodies that catalyze primary amide bond hydrolysis or for mation. We characterized a catalytic antibody, 13D11, raised to a phos phinate hapten, that hydrolyzed the primary amide of a dansyl-alkylate d derivative of (R)-phenylalaninamide (DNS-(R)F-NH2). At pH 9.0, 13D11 hydrolyzed DNS-(R)F-NH2 with a k(cat) of 1.65 x 10(-7) s(-1) (k(cat)/ k(uncat) = 132) and a K-m of 432 mu M, and was stereospecifically hapt en-inhibited (K-i = 14.0 mu M). 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 hy drolysis, the rate of DNS-(R)F-NH2 hydrolysis increased with hydroxide concentration to an optimum pH of 9.5. Above pH 9.5, activity decline d rapidly suggesting the antibody was inactivated during the long intu bation period. This work demonstrates the feasibility of generating ca talytic antibodies to hydrolyze unactivated amide bonds without cofact or assistance.