Communications
report an unconventional computational design that has led
tational modeling also suggests that the formation of the
prereactive BChE–(À)-cocaine complex (ES) is hindered
mainly by the bulky side chain of the Y332 residue in wild-
type BChE, but the hindering can be removed by the Y332A
to the discovery of a human BChE mutant with a ꢀ 151-fold
improved catalytic efficiency; this mutant can be used as an
exogenous enzyme in humans to prevent (À)-cocaine from
reaching the CNS. The encouraging outcome not only
provides a potential anticocaine medication but also demon-
strates that a novel general approach of studying enzymatic
mechanisms and computational drug design is promising.
For rational design of a mutant enzyme with a higher
catalytic activity for a given substrate, in general, one needs to
design a mutation that can accelerate the rate-determining
step of the entire catalytic reaction process while the other
steps are not slowed down by the mutation. Reported
computational modeling and experimental data indicated
that the formation of the prereactive BChE–(À)-cocaine
complex (ES) is the rate-determining step of (À)-cocaine
[
20]
or Y332G mutation.
Therefore, by starting from the
A328W/Y332A and A328W/Y332G mutants, our current
study for improving the catalytic efficiency of BChE against
(À)-cocaine aimed to decrease the energy barrier for the first
reaction step without significantly affecting the ES formation
and other chemical reaction steps. To achieve this aim,
[
25]
molecular dynamics (MD) simulations were performed to
simulate the structures of the first transition state (TS1) for
(À)-cocaine hydrolysis catalyzed by wild-type BChE and its
various mutants.
We hoped to predict some possible mutations that can
lower the energy of the TS1 structure and, therefore, lower
the energy barrier for the first reaction step. Apparently, a
mutant associated with stronger hydrogen bonding between
the carbonyl oxygen atom of (À)-cocaine benzoyl ester and
the oxyanion hole of the BChE mutant in the TS1 structure
may potentially have a more stable TS1 structure and,
therefore, a higher catalytic activity against (À)-cocaine.
Hence, hydrogen bonding with the oxyanion hole in the TS1
structure is a crucial factor affecting the transition-state
stabilization and the catalytic activity. The possible effects of
some mutations on the hydrogen bonding were examined by
performing molecular modeling and MD simulations on the
TS1 structures for (À)-cocaine hydrolysis catalyzed by wild-
type BChE and its various mutants. The initial candidate
mutants were chosen by simple geometric consideration of
the possible modification of the TS1 structure; only an energy
minimization was carried out in the simple geometric
consideration of each possible mutant. The MD simulations
were then performed only for the candidate mutants whose
energy-minimized TS1 structures clearly suggested possibly
stronger hydrogen bonding between the carbonyl oxygen
atom of (À)-cocaine and the oxyanion hole of the enzyme.
The MD simulation in water was performed for 1 ns or
longer to make sure that we obtained a stable MD trajectory
for each simulated TS1 structure with the wild-type or mutant
BChE. The MD trajectories actually became stable quickly
and so did the H···O distances involved in the potential
hydrogen bonds between the carbonyl oxygen atom of (À)-
cocaine and the oxyanion hole of BChE. The H···O distances
in the simulated TS1 structures for wild-type BChE and its
four mutants are summarized in Table 1 (see the Supporting
Information for the key MD trajectory and MD-simulated
TS1 structures).
[
17–23]
hydrolysis catalyzed by wild-type BChE,
whereas the
rate-determining step for the faster hydrolysis of the biolog-
ically inactive (+)-cocaine enantiomer is the chemical reac-
tion process consisting of four individual reaction steps
[
18]
(
Scheme 1). This mechanistic understanding is consistent
[
17]
with the experimental observation that the catalytic rate
constant of wild-type BChE against (+)-cocaine is pH-
dependent, whereas that of the same enzyme against (À)-
cocaine is independent of the pH value. The pH-dependence
of the rate constant for (+)-cocaine hydrolysis is clearly
associated with the protonation of the H438 residue in the
catalytic triad (S198, H438, and E325). For the first and third
steps of the reaction process, when H438 is protonated, the
catalytic triad cannot function and, therefore, the enzyme
becomes inactive. The lower the pH value of the reaction
solution, the higher the concentration of protonated H438
and the lower the concentration of the active enzyme. Hence,
the rate constant was found to decrease with a decreasing pH
value of the reaction solution for the enzymatic hydrolysis of
[
17]
(
+)-cocaine. Based on the above mechanistic understand-
ing, the previously reported efforts for rational design of
BChE mutants have focused on how to improve the ES
[
18,20,24]
formation process.
[
18]
Experimental observation also indicated that the cata-
lytic rate constant of A328W/Y332A BChE is pH-dependent
for both (À)- and (+)-cocaine. The pH-dependence reveals
that, for both (À)- and (+)-cocaine, the rate-determining step
of the hydrolysis catalyzed by A328W/Y332A BChE should
be either the first or the third step of the reaction process.
Further, if the third step were rate determining, then the
catalytic efficiency of the A328W/Y332A mutant against (À)-
cocaine should be as high as that of the same mutant against
(
+)-cocaine because the (À)- and (+)-cocaine hydrolyses
As seen in Table 1, in the simulated TS1 structures for the
wild-type, A328W/Y332A, A328W/Y332G, and F227A/
S287G/A328W/Y332M BChEs, the carbonyl oxygen atom of
(À)-cocaine can form up to two NÀH···O hydrogen bonds
share the same third and fourth steps (see Scheme 1).
However, it has been observed that the A328W/Y332A
mutant only has a ꢀ 9-fold improved catalytic efficiency
against (À)-cocaine, whereas the A328W/Y332A mutation
does not change the high catalytic activity against (+)-
cocaine. This analysis of the experimental and computa-
tional data available in the literature clearly shows that the
rate-determining step of (À)-cocaine hydrolysis catalyzed by
the A328W/Y332A mutant should be the first step of the
chemical reaction process. Further, recently reported compu-
with the peptidic NH hydrogen atoms of G117 and A199. The
overall strength of the hydrogen bonding between the
carbonyl oxygen atom of (À)-cocaine and the oxyanion hole
of the enzyme only slightly increases when wild-type BChE is
replaced by the A328W/Y332A, A328W/Y332G, or F227A/
S287G/A328W/Y332M mutants, as seen from the estimated
total hydrogen-binding energy (HBE) values in Table 1. In
[
18]
6
54
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2006, 45, 653 –657
Gao, Daquan
