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duced in E. coli with a variable heavy chain (V
H
) from the antibody [64]. Imidazole was
introduced into the combining site by substituting Y
34
of V
L
with His using site-di-
rected mutagenesis (see Y
34
in Figure 5.18). This His mutant Fv catalyzed the hydro-
lysis of the 7-hydroxycoumarin esters of 5-(2,4-dinitrophenyl)-aminopentanoic acid
90 000-fold faster than the reaction in the presence of 4-methyl imidazole at pH
7.8. Using that substrate, Fv(Y34H
L
) turned over at least eleven times and retained
44% of its activity. The loss of activity probably resulted from the accumulation of
the inhibitory reaction product. The mutant Fv bound
-2,4-DNP-L-lysine only six-
fold less tightly than wild-type protein, suggesting that the substituted Tyr residue
is not involved in the DNP recognition process. Compared to the imidazole-catalytic
antibody generated by tethering an imidazole chemically, a sixteen-fold greater rate
increase was observed for the reactions catalyzed by His mutant Fv protein under
similar conditions. This rate enhancement is likely due to the fewer degrees of the
freedom of the imidazole moiety inside the combining site (cf.
5.19-1
and
5.19-2
).
While these results alone indicate that the construct prepared by mutagenesis func-
tioned more efficiently than the one obtained from chemical modification, this parti-
cular example does not exemplify the most powerful feature of chemical modification,
i.e., the ability to incorporate non-natural functionality or spacers into complex protein
structures. Considerable use will probably be made of this strategy in future catalyst
design.
e
5.6
Conclusions
The examples described in this chapter reveal the breadth of methods now used to
create protein-based catalysts. Structural analysis via X-ray and NMR techniques
has proved to be critical for providing atomic structures that serve as the starting point
for design. Computer modeling has become a powerful method in both the planning
of new designs and in the interpretation of experimental results. Recombinant DNA
techniques allow chemists to make mutations or more global changes in protein scaf-
folds. Together, these tools have allowed researchers to assemble new protein-based
catalysts that can be used to probe biological systems or catalyze useful chemical trans-
formations. While these catalysts do not in general rival the efficiency of natural en-
zymes, impressive rate accelerations have been obtained in some cases. New methods
including phage display [65] and antibody production promise to provide greater ac-
cess to ligand-specific scaffold development. Peptide/protein ligation techniques offer
the possibility of increasing the scope of functionality that can be incorporated into
protein structures [66]. As the understanding of how enzymes promote chemical re-
actions with high efficiency increases, so too will the ability to design more efficient
protein-based catalysts. Given the enormous power obtained from combining chemi-
cal and genetic methods it is likely that the field of protein-based artificial enzyme
design will continue to grow in the near future.
