MECHANISTIC STUDIES OF AN ANTIBODY-CATALYZED ELIMINATION-REACTION

Antibodies elicited against the ammonium ion containing hapten, N-meth yl-N-(4-nitrobenzyl)-delta-aminovaleric acid, 3, are capable of cataly zing HF elimination from (4R,4S)-fluoro-4-(4'-nitrophenyl)butan-2-one, 1. The ammonium ion in 3 is responsible for generating a complementar y negatively charged carboxylate ion in the antibody combining site wh ich serves as a general base to abstract a proton from C-3 of 1. The b ase responsible for catalysis in one antibody was identified by affini ty labeling with H-3]-(E)-3,4-epoxy-4-(4'-nitrophenyl)-butan-2-one. Pe ptide mapping of the derivatized heavy chain identified glutamate 46 a s the carboxylate group responsible for proton abstraction. A primary kinetic isotope effect of k(H)/k(D) = 2.35 for the antibody catalyzed reaction ruled out an E1 elimination mechanism but does not differenti ate between an E2 or E1cB mechanism. The stereochemistry of proton abs traction was assessed by use of the four possible C-3 monodeuterated d iastereomers of substrate 1. Product analysis demonstrated that 43D4-3 D12 is capable of abstracting either the proR or proS proton at C-3 of 1. These mechanistic studies validate the use of hapten complementari ty as a rational design strategy for introducing precisely positioned catalytic groups in antibody combining sites.