Mimicking Enzymes with Antibodies - Artificial Enzymes - page 96

Scheme 4.4 Base-catalyzed elimination of 5-nitrobenzisoxazole (11)
to give salicyclonitrile 12 is catalyzed by antibody 34E4. Haptenic
2-aminobenzimidazolium derivative 13 induced a complementary
glutamate at H50 in the binding pocket that serves as base in the
catalytic reaction.
the aromatic residues that sandwich the planar benzimidazolium in the antibody-hap-
ten structure [42]. In addition, the high degree of positional ordering imposed by the
antibody active site likely contributes significantly to catalytic efficiency. Aromatic re-
sidues lining the pocket clamp the hapten in an orientation that allows formation of a
bidentate hydrogen bond with the side chain of the carboxylate base, Glu H50 [42]. The
catalytic carboxylate, itself locked in place by hydrogen bonds with the side chain of
Asn H58 and a fixed water molecule, is optimally situated to abstract a proton from a
benzisoxazole occupying the ligand binding slot.
Replacement of the catalytic glutamate by aspartate destabilizes the transition state
for elimination by 2.1 kcal mol -1 [39]. Because the pK a of the carboxylate in the aspar-
tate mutant is the same as in the parent antibody, loss of activity is directly attributable
to suboptimal positioning of the base relative to substrate rather than to a medium
effect. This conclusion is supported by X-ray data for the mutant showing that the
aspartate carboxyl group is a little farther from the ligand than the original glutamate
and adopts an altered orientation that is stereoelectronically less suited for proton ab-
straction [42]. The advantage the antibody derives from covalently fixing its base at the
active site is further underscored by findings that high formate and acetate concen-
trations cannot rescue the inactive variants Glu H50 Ala and Glu H50 Gly, which lack an
active site base of their own [39].
Comparison of 34E4 with a less proficient catalyst shows that merely positioning a
carboxylate in a hydrophobic binding pocket does not result in efficient general base
catalysis. Antibody 4B2, generated against cationic amidinium salt 14 (Scheme 4.5),