SUBSTITUENT EFFECTS ON AN ANTIBODY-CATALYZED HYDROLYSIS OF PHENYL-ESTERS - FURTHER EVIDENCE FOR AN ACYL-ANTIBODY INTERMEDIATE

The hydrolysis of a series of para-substituted phenyl esters (2 and 6a-d) by the monoclonal catalytic antibody NPN43C9 has been investigated. The apparent pK(a) observed in the k(cat)/K(m)-pH profiles shifts from 8.9 (p-nitrophenyl ester) up to a maximum of 9.5 (p-methylphenyl ester). A correlation of the antibody-catalyzed rates of hydrolysis of the esters versus the sigma-parameter affords a rho-value of 2.3, indicative of a hydrolytic mechanism proceeding via the attack of a neutral nitrogen nucleophile and contrasting with the low rho-value expected for general base catalyzed hydrolysis. There was an inverse solvent deuterium isotope effect on the antibody-catalyzed hydrolysis of the p-chlorophenyl ester at pH > 10. These observations are consistent with our previous proposal (Benkovic, et al. Science 1990, 250, 1135) that this antibody employs a multistep kinetic pathway which involves the formation of a covalent acyl-antibody intermediate.