COPYING NATURES MECHANISM FOR THE DECARBOXYLATION OF BETA-KETO ACIDS INTO CATALYTIC ANTIBODIES BY REACTIVE IMMUNIZATION

Reactive immunization was used to generate catalytic antibodies that u se the enamine mechanism common to the natural class I aldolase enzyme s. In order to investigate the possibility of exploiting the imine and enamine intermediates programmed into antibody catalysts by reactive immunization and the features which antibody aldolases share with natu rally evolved catalysts, we have studied their ability to catalyze the decarboxylation of structurally related beta-keto acids. Both aldolas e antibodies were shown to efficiently catalyze the decarboxylation of two hapten-related beta-keto acids with rate enhancements (k(cat)/k(u ncat)) between 4959 and 14774. Inhibition studies support the role of an essential lysine residue in the active site of the antibodies and t he formation of a cyanide accessible imine intermediate in the mechani sm. Investigation of the decarboxylation reaction of 2-{3'-(4 ''-aceta midophenyl)propyl}-acetoacetic acid, 4, to 6-(4'-acetamidophenyl)-2-he xanone, 12, in the presence of O-18-labeled water by electrospray mass spectrometry revealed obligatory incorporation of O-18 in the antibod y-catalyzed reaction consistent with decarboxylation proceeding via an imine intermediate. These studies demonstrate that reactive immunizat ion may be utilized to program in fine detail the mechanism of catalyt ic antibodies and our ability to exploit the programmed reaction coord inate for different catalytic tasks.