Chemistry Reference
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folds that have binding sites tailored for a specific organic molecule to be produced.
Figure 5.18 shows the hapten binding site of MOPC 315; an impressive number of
specific contacts between the antibody and the nitrophenyl amide containing ligand
can be seen. While the immunoglobulin-derived scaffolds are quite large, the ability to
create high-affinity substrate binding sites without numerous iterations of design
makes this an attractive approach for catalyst design.
5.5.2
Incorporation of an Imidazole Functional Group into an Antibody for Catalysis
The capability of antibodies to provide tailor-made binding sites for catalyst design has
been exploited for the preparation of hydrolytic catalysts. The antibody MOPC 315
binds substituted 2,4-dinitrophenyl-containing compounds with association constants
ranging from 5
10
6
M
-1
[63]. Imidazole was incorporated into this antibody
via a thiol group introduced by chemically modifying K
52H
in the active site via a dis-
ulfide linkage (Figure 5.19). Using a series of coumarin esters as substrates, multiple
(
>
10) hydrolytic turnovers were observed with no loss of activity. The catalytic effi-
ciency, k
cat
/K
M
, for this reaction was over 10
3
times higher than for a model reaction
employing 4-methylimidazole.
10
4
to 1
5.5.3
Comparison of Imidazole-containing Antibodies Produced by Chemical Modification and
Site-directed Mutagenesis
Since an imidazole group can be incorporated into a protein through site-directed mu-
tagenesis, such an approach was used to prepare a MOPC 315 mutant containing a
histidine residue at the corresponding site. In brief, a hybrid Fv fragment of MOPC
315 was constructed by reconstituting a recombinant variable light chain (V
L
) pro-
Figure 5.19 Introduction of imidazole into the ligand binding site of
MOPC 315 through chemical modification and site-directed mutage-
nesis.
