MECHANISTIC STUDIES OF AN ANTIBODY-CATALYZED ELIMINATION-REACTION

Antibodies elicited against the ammonium ion containing hapten, N-methyl-N-(4-nitrobenzyl)-delta-aminovaleric acid, 3, are capable of catalyzing HF elimination from (4R,4S)-fluoro-4-(4'-nitrophenyl)butan-2-one, 1. The ammonium ion in 3 is responsible for generating a complementary negatively charged carboxylate ion in the antibody combining site which serves as a general base to abstract a proton from C-3 of 1. The base responsible for catalysis in one antibody was identified by affinity labeling with [4-H-3]-(E)-3,4-epoxy-4-(4'-nitrophenyl)-butan-2-one. Peptide mapping of the derivatized heavy chain identified glutamate 46 as 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 differentiate between an E2 or E1cB mechanism. The stereochemistry of proton abstraction was assessed by use of the four possible C-3 monodeuterated diastereomers of substrate 1. Product analysis demonstrated that 43D4-3D12 is capable of abstracting either the proR or proS proton at C-3 of 1. These mechanistic studies validate the use of hapten complementarity as a rational design strategy for introducing precisely positioned catalytic groups in antibody combining sites.