Chemistry Reference
In-Depth Information
perfect shape complementarity between protein and transition state analog, replace-
ment of Met
H100b
at the floor of the pocket by phenylalanine increased the affinity for
3
by a factor of two and augmented k
cat
7-fold [33]. Even higher activity is likely to be
attainable if the affinity of the antibody for the transition state (analog) can be further
increased.
Although nanomolar affinities are generally sufficient for the purposes of the im-
mune system, they are inadequate for high catalytic activity. Enzymes are believed to
bind transition states many orders of magnitude more tightly than 1E9 binds the hex-
achloronorbornene
3
(K
d
= 0.1 n
M
[34]). In effect, with respect to catalysis, affinity
maturation stops too soon. In principle, it should be possible to continue the evolution
of these first generation catalysts in vitro by subjecting them to random mutagenesis,
and screening the resulting libraries for tighter ligand binding or directly for higher
activity. Nanomolar binders have been converted into receptors with picomolar to fem-
tomolar affinities in this way [35]. If similar effects can be achieved with 1E9, a sig-
nificant fraction of the binding energy gained may be manifest as enhanced catalytic
efficiency.
4.5
General Acid-Base Catalysis
Highly evolved enzymes often possess sophisticated arrays of functional groups that
they use to promote otherwise difficult reactions. Although catalytically useful acids,
bases, or nucleophiles can arise by chance in the immunoglobulin pocket during evo-
lution of the immune response to transition state analogs, other measures are gen-
erally needed to elicit reactive residues reliably and systematically. Charge comple-
mentarity between antibody and antigen has been extensively exploited for this pur-
pose. For example, positively charged ammonium salts have yielded various antibodies
that utilize carboxyl groups to promote eliminations, enolizations, hydrolytic reactions
and isomerizations [2].
Antibody catalysts for converting benzisoxazoles (
11
) into salicylonitriles (
12
)
(Scheme 4.4), a well-characterized E2 elimination that is sensitive to base strength
and solvent microenvironment [36], illustrate the strategic use of haptenic charge.
The cationic 2-aminobenzimidazolium derivative
13
, which mimics the overall geo-
metry of the elimination transition state and bears little resemblance to the reaction
product, was employed as a hapten [37]. Antibody 34E4, which binds
13
tightly (K
d
1n
M
), catalyzes the target reaction with more than 10
3
turnovers per active site and a
rate acceleration of 10
6
over background. A glutamate at H50 serves as the catalytic
base [38, 39]. The effective molarity for this residue is
>
10 000
M
[37, 39], an excep-
tionally high value relative to EMs obtained in intramolecular model systems for gen-
eral base catalysis [40].
The efficiency of 34E4 likely derives from a combination of factors [38, 41]. Medium
effects are certainly relevant. The carboxylate base is activated by placement in a re-
latively hydrophobic environment, as evidenced by its elevated pK
a
of 6 [37]. The charge
delocalized transition state is probably also stabilized by dispersive interactions with
