Chemistry Reference
In-Depth Information
The germline precursor of 48G7 was isolated from the primary immunological
repertoire [14]. It also accelerates ester hydrolysis, yet is 20-fold less efficient than
the mature antibody and binds the hapten 30 000-fold less tightly. During affinity ma-
turation, nine somatic mutations were introduced into the antibody scaffold. Although
none of the altered residues directly contacts bound
1
, the substitutions appear to
organize the pocket for binding and catalysis by limiting side-chain and backbone flex-
ibility inherent in the germline protein [15]. Thus, ligand binding to germline 48G7
induces significant structural reorganization, whereas the free and ligand-bound
forms of the mature antibody are very similar. The germline and mature antibodies
also display some differences in the way they recognize the hapten and, by extension,
the hydrolytic transition state. For example, because of an altered conformation for the
CDR H1 loop, the aryl leaving group adopts different orientations at the two active
sites. More importantly, due to conformational constraints, Tyr
H33
cannot provide a
hydrogen bond to the anionic transition state in the germline complex, resulting
in reduced catalytic efficiency.
Parallel evolution of hapten binding and catalytic activity has been observed in sev-
eral catalytic antibodies in addition to 43G7 [8, 16], illustrating the value of immuniza-
tion as a means of optimizing the catalytic potential of the antibody combining site.
Nevertheless, the correlation between binding and catalysis is necessarily approximate
because stable analogs can never perfectly mimic the stereoelectronic features of a
high energy transition state. Sometimes affinity maturation even causes a loss of ac-
tivity, as seen in antibody AZ-28 [17], which catalyzes an oxy-Cope rearrangement.
Mature AZ-28 binds the transition state analog 40-times more tightly than its germline
precursor, but it is 30-fold less efficient as a catalyst. This inverse relationship has been
attributed to structural differences between the transition state analog and the transi-
tion state, and differences in the flexibility of the mature and germline antibodies [18].
Because only a fraction of the antibodies induced in response to any given transition
state analog typically exhibits catalytic activity, screening is an essential step in any
catalytic antibody experiment. Consequently, much effort has been invested in the
development of sensitive high-throughput assays [19]. For the same reason, access
to large and diverse populations of hapten-binders is desirable. With respect to the
latter point, autoimmune mice yield unusually large numbers of esterolytic antibodies
[20], and may usefully expand the repertoire of catalytic clones elicited by other transi-
tion state analogues.
4.4
Importance of a Good Fit
The ability of enzymes to preorganize reactants for reaction contributes significantly to
their efficacy [21]. The excellent shape complementarity observed in antibody-antigen
complexes suggests that antibody binding energy could be exploited for this purpose.
Antibody catalysts have been developed for bimolecular Diels-Alder reactions to test
this supposition.
