Most efficient cocaine hydrolase designed by virtual screening of transition states

Article abstract of DOI:10.1021/ja803646t

Cocaine is recognized as the most reinforcing of all drugs of abuse. There is no anticocaine medication available. The disastrous medical and social consequences of cocaine addiction have made the development of an anticocaine medication a high priority. It has been recognized that an ideal anticocaine medication is one that accelerates cocaine metabolism producing biologically inactive metabolites via a route similar to the primary cocaine-metabolizing pathway, i.e., cocaine hydrolysis catalyzed by plasma enzyme butyrylcholinesterase (BChE). However, wild-type BChE has a low catalytic efficiency against the abused cocaine. Design of a high-activity enzyme mutant is extremely challenging, particularly when the chemical reaction process is rate-determining for the enzymatic reaction. Here we report the design and discovery of a high-activity mutant of human BChE by using a novel, systematic computational design approach based on transition-state simulations and activation energy calculations. The novel computational design approach has led to discovery of the most efficient cocaine hydrolase, i.e., a human BChE mutant with an ～2000-fold improved catalytic efficiency, promising for therapeutic treatment of cocaine overdose and addiction as an exogenous enzyme in human. The encouraging discovery resulted from the computational design not only provides a promising anticocaine medication but also demonstrates that the novel, generally applicable computational design approach is promising for rational enzyme redesign and drug discovery.

Full text of DOI:10.1021/ja803646t

Published on Web 08/19/2008
Most Efficient Cocaine Hydrolase Designed by Virtual
Screening of Transition States
Fang Zheng,† Wenchao Yang,† Mei-Chuan Ko,‡ Junjun Liu,† Hoon Cho,†
Daquan Gao,† Min Tong,† Hsin-Hsiung Tai,† James H. Woods,‡ and
Chang-Guo Zhan*,†
Department of Pharmaceutical Sciences, College of Pharmacy, UniVersity of Kentucky, 725 Rose
Street, Lexington, Kentucky 40536, and Department of Pharmacology, 1301 Medical Sciences
Research Building III, UniVersity of Michigan Medical School, Ann Arbor, Michigan 48109
Received May 16, 2008; E-mail: zhan@uky.edu
Abstract: Cocaine is recognized as the most reinforcing of all drugs of abuse. There is no anticocaine
medication available. The disastrous medical and social consequences of cocaine addiction have made
the development of an anticocaine medication a high priority. It has been recognized that an ideal anticocaine
medication is one that accelerates cocaine metabolism producing biologically inactive metabolites via a
route similar to the primary cocaine-metabolizing pathway, i.e., cocaine hydrolysis catalyzed by plasma
enzyme butyrylcholinesterase (BChE). However, wild-type BChE has a low catalytic efficiency against the
abused cocaine. Design of a high-activity enzyme mutant is extremely challenging, particularly when the
chemical reaction process is rate-determining for the enzymatic reaction. Here we report the design and
discovery of a high-activity mutant of human BChE by using a novel, systematic computational design
approach based on transition-state simulations and activation energy calculations. The novel computational
design approach has led to discovery of the most efficient cocaine hydrolase, i.e., a human BChE mutant
with an ∼2000-fold improved catalytic efficiency, promising for therapeutic treatment of cocaine overdose
and addiction as an exogenous enzyme in human. The encouraging discovery resulted from the
computational design not only provides a promising anticocaine medication but also demonstrates that the
novel, generally applicable computational design approach is promising for rational enzyme redesign and
drug discovery.
Introduction
encouraging outcome in vitro and in vivo not only provides a
promising anticocaine medication but also demonstrates a novel,
Cocaine is recognized as the most reinforcing of all drugs of
abuse.1-3 There is no anticocaine medication available. The
disastrous medical and social consequences of cocaine addiction
have made the development of an anticocaine medication a high
priority.4,5 An ideal anticocaine medication would be to ac-
celerate cocaine metabolism producing biologically inactive
metabolites via a route similar to the primary cocaine-
metabolizing pathway, i.e., cocaine hydrolysis catalyzed by
plasma enzyme butyrylcholinesterase (BChE).4,6-10 However,
wild-type BChE has a low catalytic efficiency against naturally
occurring (-)-cocaine.11-15 Here we report a unique compu-
tational design leading to discovery of a human BChE mutant
with an ∼2000-fold improved catalytic efficiency, which is
sufficient for use as an exogenous enzyme in human to prevent
(-)-cocaine reaching central nervous system (CNS). The
generally applicable approach for rational enzyme redesign and
drug discovery.
The primary pathway for cocaine metabolism in primates is
hydrolysis at the benzoyl ester or methyl ester group.4,10 Benzoyl
ester hydrolysis generates ecgonine methyl ester (EME), whereas
the methyl ester hydrolysis yields benzoylecgonine (BE). The
major cocaine-metabolizing enzymes in humans are BChE
which catalyzes benzoyl ester hydrolysis (Figure 1) and two
liver carboxylesterases (denoted by hCE-1 and hCE-2) which
(7) Carrera, M. R. A.; Kaufmann, G. F.; Mee, J. M.; Meijler, M. M.;
Koob, G. F.; Janda, K. D. Proc. Natl. Acad. Sci. U.S.A. 2004, 101,
10416–10421.
(8) Landry, D. W.; Zhao, K.; Yang, G. X.-Q.; Glickman, M.; Georgiadis,
T. M. Science 1993, 259, 1899–1901.
(9) Zhan, C.-G.; Deng, S.-X.; Skiba, J. G.; Hayes, B. A.; Tschampel, S. M.;
Shields, G. C.; Landry, D. W. J. Comput. Chem. 2005, 26, 980–986.
(10) Kamendulis, L. M.; Brzezinski, M. R.; Pindel, E. V.; Bosron, W. F.;
Dean, R. A. J. Pharmacol. Exp. Ther. 1996, 279, 713–717.
(11) Sun, H.; Pang, Y. P.; Lockridge, O.; Brimijoin, S. Mol. Pharmacol.
2002, 62, 220–224.
† University of Kentucky.
‡ University of Michigan Medical School.
(1) Mendelson, J. H.; Mello, N. K. N. Engl. J. Med. 1996, 334, 965–972.
(2) Singh, S. Chem. ReV. 2000, 100, 925–1024.
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109, 4776–4782.
(3) Paula, S.; Tabet, M. R.; Farr, C. D.; Norman, A. B.; Ball, W. J., Jr
J. Med. Chem. 2004, 47, 133–142.
(13) Gateley, S. J. Biochem. Pharmacol. 1991, 41, 1249–1254.
(14) Darvesh, S.; Hopkins, D. A.; Geula, C. Nat. ReV. Neurosci. 2003, 4,
131–138.
(4) Gorelick, D. A. Drug Alcohol Depend. 1997, 48, 159–165.
(5) Redish, A. D. Science 2004, 306, 1944–1947.
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Moss, J. A. J. Am. Chem. Soc. 2005, 127, 2477–2484.
(15) Giacobini, E., Ed. Butyrylcholinesterase: Its Function and Inhibitors;
Dunitz Martin Ltd.: Great Britain, 2003.
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12148 J. AM. CHEM. SOC. 2008, 130, 12148–12155
10.1021/ja803646t CCC: $40.75
2008 American Chemical Society

Most Efficient Cocaine Hydrolase
A R T I C L E S
seizure threshold, similar to cocaine itself. Norcocaine is
hepatotoxic and a local anesthetic.17 Thus, hydrolysis of cocaine
at the benzoyl ester by an enzyme is the pathway most suit-
able for amplification. The use of an exogenous enzyme has
some potential advantages over active immunization since the
enzyme administration would immediately enhance cocaine
metabolism and would not require an immune response to be
effective.4
Ignoring hCE-2, so far, two types of known native enzymes
may be used to catalyze hydrolysis of cocaine at the benzoyl
ester: human BChE and bacterial cocaine esterase (CocE).18,19
BChE from human source can be tolerated perfectly in the
human body, and in fact, BChE has a long history of successful
clinic use for other purposes.4 However, the catalytic activity
of this plasma enzyme (kcat ) 4.1 min-1 and KM ) 4.5 µM)11
is significantly lower against the naturally occurring (-)-cocaine
than CocE (kcat ) 468 min-1 and KM ) 0.64 µM).19 Due to the
low activity of BChE, (-)-cocaine has a plasma half-life of
∼45-90 min (before BChE saturation), long enough for
manifestation of the effects on CNS which peak in minutes.13
Hence, BChE mutants with a significantly higher activity against
(-)-cocaine are highly desired for use as an exogenous enzyme
in humans.
In general, for rational design of an enzyme mutant with a
higher catalytic activity for a given substrate, 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-(-)-cocaine complex (ES) is the rate-
determining step of (-)-cocaine hydrolysis (Figure 1) catalyzed
by wild-type BChE,11,12,20,21 whereas the rate-determining step
of the unnatural, biologically inactive (+)-cocaine hydrolysis
catalyzed by the same enzyme is the chemical reaction process
consisting of four individual reaction steps.21 On the basis of
this mechanistic understanding, previous efforts reported by
other researchers for rational design of BChE mutants were
focused on how to improve the ES formation process, and
several BChE mutants11,12,22 were found to have an ∼9-34-
fold improved catalytic efficiency (kcat/KM) against (-)-cocaine.
Recently reported computational modeling also suggests that
the formation of the prereactive BChE-(-)-cocaine complex
(ES) is hindered mainly by the bulky side chain of Y332 residue
in wild-type BChE, but the hindering can be removed by the
Y332A mutation, and the Y332G mutation can produce a more
significant improvement.12 The combined computational and
experimental data11,20,23-26 have revealed that the rate-determin-
(16) Poet, T. S.; McQueen, C. A.; Halpert, J. R. Drug Metab. Dispos. 1996,
24, 74–80.
(17) Pan, W.-J.; Hedaya, M. A. J. Pharm. Sci. 1990, 88, 468–476.
(18) Larsen, N. A.; Turner, J. M.; Stevens, J.; Rosser, S. J.; Basran, A.;
Lerner, R. A.; Bruce, N. C.; Wilson, I. A. Nat. Struct. Biol. 2002, 9,
17–21.
Figure 1. Hydrolysis reactions of (-)-cocaine and (+)-cocaine: (A)
reactants and products of the reactions; (B) schematic representation of the
first reaction step for (-)-cocaine hydrolysis catalyzed by a BChE mutant
including A199S mutation.
(19) Turner, J. M.; Larsen, N. A.; Basran, A.; Barbas, C. F.; Bruce, N. C.;
Wilson, I. A.; Lerner, R. A. Biochemistry 2002, 41, 12297–12307.
(20) Sun, H.; Yazal, J. E.; Lockridge, O.; Schopfer, L. M.; Brimijoin, S.;
Pang, Y. P. J. Biol. Chem. 2001, 276, 9330–9336.
catalyze hydrolysis at the methyl ester and the benzoyl ester,
respectively. Among the three, BChE is the principal cocaine
hydrolase in human serum. Hydrolysis accounts for about 95%
of cocaine metabolism in humans. The remaining 5% is
deactivated through oxidation by the liver microsomal cyto-
chrome P450 system, producing norcocaine.4,16 EME appears
the least pharmacologically active of the cocaine metabolites
and may even cause vasodilation,4 whereas both BE and
norcocaine appear to cause vasoconstriction and lower the
(21) Zhan, C.-G.; Zheng, F.; Landry, D. W. J. Am. Chem. Soc. 2003, 125,
2462–2474.
(22) Gao, Y.; Atanasova, E.; Sui, N.; Pancook, J. D.; Watkins, J. D.;
Brimijoin, S. Mol. Pharmacol. 2005, 67, 204–211.
(23) Zhan, C.-G.; Gao, D. Biophys. J. 2005, 89, 3863–3872.
(24) Gao, D.; Zhan, C.-G. J. Phys. Chem. B 2005, 109, 23070–23076.
(25) Pan, Y.; Gao, D.; Yang, W.; Cho, H.; Yang, G.-F.; Tai, H.-H.; Zhan,
C.-G. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 16656–16661.
(26) Gao, D.; Zhan, C.-G. Proteins 2006, 62, 99–110.
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A R T I C L E S
Zheng et al.
screening of various BChE mutants. The virtual screening starts
by first estimating the total interaction energy (TIE) between
the whole oxyanion hole of the enzyme and all (-)-cocaine
atoms in the TS1 structure for each possible mutant. The
practical effects of the interactions on the energy barrier for
the rate-determining reaction step are evaluated by performing
more sophisticated, hybrid quantum mechanical/molecular me-
chanical (QM/MM) calculations, followed by wet experimental
tests in vitro and in vivo.
Results and Discussion
To carry out the TIE-based initial screening, we first examined
a variety of possible BChE mutants by performing molecular
dynamics (MD) simulation on the TS1 structure for each mutant.
The general strategy of performing a classical MD simulation
on a transition-state structure of the enzymatic reaction has been
described and justified in our recently reported studies.23-26,28
Each MD simulation in water was performed for 1 ns or longer
to make sure that we obtained a stable MD trajectory for each
TS1 structure simulated. Within the stable MD trajectory
obtained for a dynamically stable TS1 structure, we collected a
large number of snapshots of the simulated TS1 structure (one
snapshot for each 1 ps). One can estimate a TIE value for each
snapshot of the simulated TS1 structure. In principle, a TIE value
includes energetic contributions from both the hydrogen-bonding
and nonbonding interactions. Technically, based on the popularly
used Amber force field,30 the TIE value can be evaluated as
the sum of the electrostatic and van der Waals interaction
energies between all atoms of (-)-cocaine and all atoms of
residue nos. 116, 117, and 199. The final TIE value is the
average of the TIE values calculated for all of the snapshots of
the MD-simulated TS1 structure.
The virtual screening based on extensive transition-state
simulations and subsequent TIE calculations reveals that the
most stable TS1 structure is associated with the A199S/F227A/
S287G/A328W/Y332G mutant. Summarized in Table 1 are
important numerical results concerning the MD-simulated TS1
structures and energetics for wild-type BChE and for representa-
tive mutants of BChE. The simulated H · · ·O distance D1 (Table
1) is always too long for the peptidic NH of G116 to form an
N-H· · ·O hydrogen bond with the carbonyl oxygen of (-)-
cocaine in all of the simulated TS1 structures. The interaction
energy (IE116) of all (-)-cocaine atoms with all atoms of G116
is purely nonbonding, as seen in Table 1. All of the calculated
IE116 values are positive, except for the A199S/F227A/S287G/
A328W/Y332G mutant. The positive interaction energy (IE)
value means to destabilize the TS1 structure, whereas the
negative IE value means to stabilize the TS1 structure. In the
simulated TS1 structure for wild-type BChE, the carbonyl
oxygen of (-)-cocaine formed an N-H· · ·O hydrogen bond
with the peptidic NH hydrogen atom of A199 residue; the
simulated H · · ·O distance (D3) was 1.61-2.35 Å, with an
average D3 value of 1.92 Å. Meanwhile, the carbonyl oxygen
of (-)-cocaine also had a partial N-H· · ·O hydrogen bond with
the peptidic NH hydrogen atom of G117 residue; the simulated
H· · ·O distance (D2) was 1.97-4.14 Å (the average D2 value:
2.91 Å). Due to the hydrogen bonding, all of the calculated
interaction energies IE117 and IE199 (including both the
hydrogen-bonding and nonbonding interaction energies) in Table
1 are negative values.
Figure 2. Schematic representation of structure- and mechanism-based
computational design and discovery of high-activity mutants of human
BChE. TIE refers to the total interaction energy between the whole oxyanion
hole of the enzyme and all (-)-cocaine atoms in the TS1 structure for each
possible mutant. ∆Eq represents the energy barrier for the first step of the
enzymatic hydrolysis of (-)-cocaine. The approach depicted here can be
used for any other enzyme, as TIE can be the total interaction energy
between all (or some key) atoms of the substrate and all (or some key)
atoms of the enzyme.
ing step of (-)-cocaine hydrolysis catalyzed by the A328W/
Y332A and A328W/Y332G mutants is the first step of the
chemical reaction process. Therefore, starting from the A328W/
Y332A or A328W/Y332G mutant, our work for further improv-
ing the catalytic efficiency of BChE against (-)-cocaine aimed
to decrease the energy barrier for the first reaction step without
significantly affecting the ES formation and other chemical
reaction steps.23,25,27,28
Our recently reported computational studies suggest that the
hydrogen bonding between the carbonyl oxygen of (-)-cocaine
benzoyl ester and the oxyanion hole (residue nos. 116, 117,
and 199) of the enzyme in the rate-determining transition state
(TS1, i.e., the transition state for the first step of the chemical
reaction process) can be enhanced by some mutations.23,25,26
The enhanced hydrogen bonding (HB) may potentially stabilize
the TS1 structure and thus possibly lower the energy barrier
for the first reaction step of (-)-cocaine hydrolysis. The HB
energy-based virtual screening led to discovery of some high-
activity mutants of BChE; the most active one is A199S/S287G/
A328W/Y332G BChE25 whose catalytic efficiency (kcat/KM)
against (-)-cocaine is still slightly lower than bacterial CocE.
A199S/S287G/A328W/Y332G BChE can selectively block
cocaine toxicity and reinstatement of drug seeking in rats.29
Concerning the computational design methodology, our
previous HB energy calculations23,25,26 were simply based on
an empirical HB energy equation which does not account for
any nonbonding interaction. So, nonbonding interactions be-
tween the (-)-cocaine atoms and the oxyanion hole of the
enzyme and their effects on the energy barrier of the enzymatic
reaction remain to be evaluated in computational modeling. In
the present study, we first aimed to develop and test a novel,
systematic design protocol (depicted in Figure 2) for a virtual
(27) Gao, D.; Cho, H.; Yang, W.; Pan, Y.; Yang, G.-F.; Tai, H.-H.; Zhan,
C.-G. Angew. Chem., Int. Ed. 2006, 45, 653–657.
(28) Pan, Y.; Gao, D.; Yang, W.; Cho, H.; Zhan, C.-G. J. Am. Chem. Soc.
2007, 129, 13537–13543.
(29) Brimijoin, S.; Gao, Y.; Anker, J. J.; Gliddon, L. A.; LaFleur, D.; Shah,
R.; Zhao, Q.; Singh, M.; Carroll, M. E. A. Neuropsychopharmacology
[Online early access]. DOI: 10.1038/sj.npp.1301666. Published Online:
January 16, 2008.
(30) Case, D. A.; et al. Amber8; University of California: San Francisco,
CA, 2004.
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A R T I C L E S
Table 1. MD-Simulated Key Distances and the Calculated Interaction Energies (IEs) between All Atoms of (-)-Cocaine and All Atoms of the
Oxyanion Hole (Residue Nos. 116, 117, and 199) in the Simulated First Transition State (TS1)
distances (D1-D4) and interaction energies (IEs)a, b
transition state
D1 or IE116
D2 or IE117
D3/D4 or IE199
total IE (i.e., TIE)c
TS1 structure for
av (Å)
4.59
2.91
1.92
(-)-cocaine hydrolysis
catalyzed by wild-type
BChE
max (Å)
min (Å)
fluctuation (Å)
IE (kcal/mol)
av (Å)
5.73
3.35
0.35
3.18
3.62
4.14
1.97
0.35
-4.52
2.35
2.35
1.61
0.12
-4.23
1.95
-5.57
-6.44
-7.57
TS1 structure for
(-)-cocaine hydrolysis
catalyzed by A328W/
Y332A mutant of BChE
max (Å)
min (Å)
fluctuation (Å)
IE (kcal/mol)
av (Å)
4.35
2.92
0.23
0.84
3.60
3.37
1.78
0.27
-4.25
2.25
3.02
1.61
0.17
-3.03
1.97
TS1 structure for
(-)-cocaine hydrolysis
catalyzed by A328W/
Y332G mutant of BChE
max (Å)
min (Å)
fluctuation (Å)
IE (kcal/mol)
av (Å)
4.24
2.89
0.23
0.04
4.39
3.17
1.77
0.24
-4.94
2.60
2.76
1.62
0.17
-2.67
2.01/1.76
TS1 structure for
(-)-cocaine hydrolysis
catalyzed by A199S/
S287G/A328W/Y332G
mutant of BChE
max (Å)
min (Å)
fluctuation (Å)
IE (kcal/mol)
av (Å)
5.72
2.87
0.48
2.96
5.28
4.42
1.76
0.36
-5.12
2.15
2.68/2.50
1.62/1.48
0.17/0.12
-9.92
-12.08
-14.78
TS1 structure for
1.96/2.20
(-)-cocaine hydrolysis
catalyzed by A199S/
F227A/S287G/A328W/
Y332G mutant of BChE
max (Å)
min (Å)
fluctuation (Å)
IE (kcal/mol)
5.98
2.72
0.25
2.83
1.68
0.18
2.40/3.28
1.65/1.59
0.12/0.30
-8.22
-0.97
-5.59
a D1, D2, and D3 represent the internuclear distances between the carbonyl oxygen of cocaine benzoyl ester and the NH hydrogen of residue nos.
116 (i.e., G116), 117 (i.e., G117), and 199 (i.e., A199 or S199) of BChE, respectively. D4 is the internuclear distance between the carbonyl oxygen of
cocaine benzoyl ester and the hydroxyl hydrogen of the S199 side chain in the A199S/S287G/A328W/Y332G and A199S/F227A/S287G/A328W/Y332G
mutants. b IE116, IE117, and IE199 represent the average interaction energies (IEs) between all atoms of (-)-cocaine and all atoms of amino acid
residue nos. 116, 117, and 199 of BChE, respectively, in the MD-simulated TS1 structure. c TIE ) IE116 + IE117 + IE199.
In going from wild-type BChE to the A328W/Y332A and
A328W/Y332G mutants, the largest IE change in the TS1
structure is associated with the decrease of the IE116 value, as
seen in Table 1. Hence, the total IE (i.e., TIE ) IE116 + IE117
+ IE199) value between all atoms of (-)-cocaine and all atoms
of the oxyanion hole only slightly decreases when wild-type
BChE is replaced by the A328W/Y332A or A328W/Y332G
mutant, as seen from the calculated TIE values in Table 1. In
the simulated TS1 structures for A199S/S287G/A328W/Y332G
and A199S/F227A/S287G/A328W/Y332G BChE’s, an O-H· · ·O
hydrogen bond formed between the hydroxyl group on the S199
side chain and the carbonyl oxygen of (-)-cocaine, in addition
to the two N-H· · ·O hydrogen bonds with the peptidic NH of
G117 and S199, as seen in Table 1. Due to the additional
O-H· · ·O hydrogen bond with the hydroxyl oxygen of residue
no. 199, the IE199 values calculated for A199S/S287G/A328W/
Y332G and A199S/F227A/S287G/A328W/Y332G BChE’s are
remarkably lower than those calculated for wild-type, A328W/
Y332A, and A328W/Y332G BChE’s, as seen in Table 1. Thus,
the TIE values calculated for the A199S/S287G/A328W/Y332G
and A199S/F227A/S287G/A328W/Y332G mutants are remark-
ably lower than the TIE values for wild-type, A328W/Y332A,
and A328W/Y332G BChE’s. The lowest TIE value is associated
with the A199S/F227A/S287G/A328W/Y332G mutant, due to
the contributions from both the additional hydrogen bond with
S199 side chain and the nonbonding interactions with residue
G116.
The calculated relative TIE values clearly suggest that the
MD-simulated TS1 structure for (-)-cocaine hydrolysis cata-
lyzed by A199S/F227A/S287G/A328W/Y332G BChE should
be more stable than the TS1 structures corresponding to wild-
type BChE and other mutants. To examine whether the TS1
stabilization can decrease the energy barrier for the rate-
determining reaction step, we further performed QM/MM
reaction coordinate calculations (at the B3LYP/6-31G*:Amber
level) on the first step of (-)-cocaine hydrolysis catalyzed by
the A199S/A328W and A199S/F227A/S287G/A328W/Y332G
mutants. The QM/MM calculations were performed to optimize
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A R T I C L E S
Zheng et al.
TIE decrease of ∼8 kcal/mol estimated from the initial IE
calculations. The QM/MM calculations qualitatively support the
results obtained from the TIE-based virtual screening. All of
the computational results consistently suggest that A199S/
F227A/S287G/A328W/Y332G BChE should be a very active
cocaine hydrolase catalyzing hydrolysis of (-)-cocaine.
To examine the theoretical prediction of high catalytic
efficiency for A199S/F227A/S287G/A328W/Y332G BChE
against (-)-cocaine, we produced the A199S/F227A/S287G/
A328W/Y332G mutant through site-directed mutagenesis, pro-
tein expression, and enzyme activity assays in vitro and in vivo.
To minimize the possible systematic experimental errors of the
in vitro kinetic data, we expressed the enzymes and performed
kinetic studies with wild-type BChE and the A199S/F227A/
S287G/A328W/Y332G mutant under the same condition and
compared the catalytic efficiency of A199S/F227A/S287G/
A328W/Y332G BChE to that of wild-type BChE for (-)-
cocaine hydrolysis at benzoyl ester group. Michaelis-Menten
kinetics of the enzymatic (-)-cocaine hydrolysis at benzoyl ester
group was determined by performing the sensitive radiometric
assays using [3H](-)-cocaine (labeled on its benzene ring) with
varying concentrations of the substrate in combination with an
enzyme-linked immunosorbent assay (ELISA). The kinetic
analysis revealed that kcat ) 5700 ( 400 min-1 and KM ) 3.1
( 0.2 µM for (-)-cocaine hydrolysis catalyzed by A199S/
F227A/S287G/A328W/Y332G BChE while the known kinetic
parameters (kcat ) 4.1 min-1 and KM ) 4.5 µM)11 for (-)-
cocaine hydrolysis catalyzed by wild-type BChE were repro-
duced by the same in vitro activity assays. Thus, A199S/F227A/
S287G/A328W/Y332G BChE has an ∼2020-fold improved
catalytic efficiency (kcat/KM ) ∼1.84 × 109 M min-1) compared
to wild-type BChE (kcat/KM ) ∼9.1 × 105 M min-1) against
(-)-cocaine.
What does the ∼2020-fold improvement of the catalytic
efficiency mean for decreasing the (-)-cocaine half-life in
plasma? With the use of the designed A199S/F227A/S287G/
A328W/Y332G BChE as an exogenous enzyme in human, when
the concentration of this mutant is kept the same as that of wild-
type BChE in plasma, the half-life of (-)-cocaine in plasma
should be reduced from the ∼45-90 min to only ∼1.3-2.7 s.
It is interesting to note that the catalytic efficiency of A199S/
F227A/S287G/A328W/Y332G BChE is even significantly higher
than that of the most active native enzyme yet identified, i.e.,
bacterial CocE with kcat/KM ) ∼7.2 × 108 M min-1, against
(-)-cocaine. More importantly, the catalytic rate constant (kcat
) 5700 min-1) of A199S/F227A/S287G/A328W/Y332G BChE
Figure 3. Results from the QM/MM reaction coordinate calculations at
the B3LYP/6-31G*:Amber level. (A) Optimized geometry of the transition
state (TS1) for the first reaction step of (-)-cocaine hydrolysis catalyzed
by A328W/Y332G BChE. (B) Optimized geometry of the transition state
(TS1) for the first reaction step of (-)-cocaine hydrolysis catalyzed by
A199S/F227A/S287G/A328W/Y332G BChE. (C) Calculated potential
energy surface along the reaction coordinate (R1 - R2 + R3) of the
enzymatic hydrolysis of (-)-cocaine. R1 is the length of the C · · ·O transition
bond between the hydroxyl oxygen of S198 and carbonyl carbon of (-)-
cocaine benzoyl ester. R2 refers to the length of the O · · ·H transition bond
in the S198 hydroxyl group. R3 represents the length of the N · · ·H transition
bond between the hydroxyl hydrogen of S198 and the nitrogen of H438
side chain.
against (-)-cocaine is higher than that (kcat ) 468 min-1 19
bacterial CocE by ∼12-fold.
)
of
Assessment of the catalytic activity using an in vivo model
based on protection of A199S/F227A/S287G/A328W/Y332G
BChE against cocaine-induced lethality correlated well with the
in vitro activity data. Our in vivo activity assays on the BChE
mutant were performed to compare with the in vivo activity of
CocE under the same experimental condition. It has been
demonstrated31 that the minimum effective dose of CocE to
protect mice from cocaine-induced lethality is 0.1 mg (per
mouse). Our theoretical estimation of the minimum effective
dose of A199S/F227A/S287G/A328W/Y332G BChE to protect
mice from cocaine-induced lethality was based on the relative
the ES, TS1, and INT1 geometries and determine the corre-
sponding energy changes from ES to TS1 and INT1. Depicted
in Figure 3 are the QM/MM-optimized TS1 geometries and the
calculated energy profiles along the reaction coordinate for the
(-)-cocaine hydrolysis catalyzed by the two mutants. As seen
in Figure 3C, the energy barrier calculated for the first step of
(-)-cocaine hydrolysis catalyzed by A199S/A328W BChE is
∼16.2 kcal/mol, whereas the energy barrier calculated for the
first step of (-)-cocaine hydrolysis catalyzed by A199S/F227A/
S287G/A328W/Y332G BChE is only ∼10.4 kcal/mol. Accord-
ing to the QM/MM results, the energy barrier decreases by ∼6
kcal/mol in going from A199S/A328W BChE to A199S/F227A/
S287G/A328W/Y332G BChE. The QM/MM-calculated energy
barrier decrease of ∼6 kcal/mol correlates very well with the
(31) Ko, M.-C.; Bowen, L. D.; Narasimhan, D.; Berlin, A. A.; Lukacs,
N. W.; Sunahara, R. K.; Cooper, Z. D.; Woods, J. H. J. Pharmacol.
Exp. Ther. 2007, 320, 926–933.
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Most Efficient Cocaine Hydrolase
A R T I C L E S
determining transition state and calculate the energy barriers
associated with the wild-type and mutant enzymes. The TIE to
be calculated in virtual screening step no. 2 can be the total
interaction energy between all (or some key) atoms of the
substrate and all (or some key) atoms of the enzyme. For
example, the similar computational design strategy integrated
with appropriate wet experiments may also be valuable in
rational redesign of other metabolic enzymes for therapeutic
treatment of metabolic diseases or for detoxification of toxic
compounds (including chemical warfare nerve agents).
Conclusion
We have developed a novel, generally applicable computa-
tional design approach based on a systematic virtual screening
of transition states of enzymatic reaction and activation energy
calculations. The novel design approach has led to discovery
of a human BChE mutant with an ∼2000-fold improved
catalytic efficiency, sufficient for therapeutic use as an exog-
enous enzyme in human to treat cocaine overdose and addiction.
The encouraging discovery resulted from the computational
design not only provides a promising anticocaine medication
but also demonstrates that the novel, generally applicable
computational design approach is promising for rational enzyme
redesign and drug discovery.
Figure 4. Protective effects of A199S/F227A/S287G/A328W/Y332G
BChE against cocaine-induced toxicity. The BChE mutant (mg/mouse) was
administered intravenously 1 min before intraperitoneal administration of
cocaine 180 mg/kg. Each data point represents the percentage of mice (n
) 6 for each dosing condition) exhibiting cocaine-induced convulsions or
lethality. The asterisks represent a significant difference from the condition
of mice pretreated with phosphate-buffered saline (p < 0.05). See the
Supporting Information for other details.
Materials and Methods
1. Molecular Dynamics Simulations. The general strategy of
performing a classical MD simulation on a transition-state structure
of an enzymatic reaction has been described and justified in our
recent studies.23-26 In principle, MD simulation using a classical
force field (molecular mechanics) can only simulate a stable
structure corresponding to a local minimum on the potential energy
surface, whereas a transition state during a reaction process is
always associated with a first-order saddle point on the potential
energy surface. Hence, MD simulation using a classical force field
cannot directly simulate a transition state without any restraint on
the geometry of the transition state. Nevertheless, if we can
technically remove the freedom of imaginary vibration in the
transition-state structure, then the number of the degrees of
vibrational freedom (normal vibration modes) for a nonlinear
molecule will decrease from 3N - 6. The transition-state structure
is associated with a local minimum on the potential energy surface
within a subspace of the reduced degrees of vibrational freedom,
although it is associated with a first-order saddle point on the
potential energy surface with all of the 3N - 6 degrees of vibrational
freedom. Theoretically, the degree of vibrational freedom associated
with the imaginary vibrational frequency in the transition-state
structure can be removed by appropriately freezing the reaction
coordinate. The reaction coordinate corresponding to the imaginary
vibration of the transition state is generally characterized by a
combination of some key geometric parameters. These key geo-
metric parameters are bond lengths of the transition bonds, i.e.,
the forming and breaking covalent bonds during this reaction step
of BChE-catalyzed hydrolysis of cocaine, as seen in Figure 1 of
the text. Thus, we just need to maintain the lengths of the transition
bonds during the MD simulation on a transition state. Technically,
we can maintain the lengths of the transition bonds by simply fixing
all atoms within the reaction center, by using some constraints on
the transition bonds, or by redefining the transition bonds. It should
be pointed out that the only purpose of performing such type of
MD simulation on a transition state is to examine the dynamic
change of the protein environment surrounding the reaction center
and the interaction between the reaction center and the protein
environment. We are only interested in the simulated structures,
as the total energies calculated in this way are meaningless.
The initial BChE structures used in the MD simulations were
prepared based on our previous MD simulation12 on the prereactive
kcat values of CocE and A199S/F227A/S287G/A328W/Y332G
BChE (kcat ) 468 min-1 for CocE versus kcat ) 5700 min-1
for the BChE mutant) and the relative molecular weights (∼65
kDa for CocE versus ∼84 kDa for the BChE mutant). This is
because in the treatment of cocaine overdose, the cocaine
concentration in the body is significantly higher than KM of the
enzyme and the overall velocity (V) of cocaine hydrolysis is
expected to reach the maximum (Vmax), i.e., Vmax ) [E]kcat in
which [E] is the concentration of the enzyme. On the basis of
the relative molecular weights and the relative catalytic rate
constants, the minimum effective dose of A199S/F227A/S287G/
A328W/Y332G BChE to protect mice from cocaine-induced
lethality was theoretically estimated to be 0.01 mg. Figure 4
shows that the A199S/F227A/S287G/A328W/Y332G mutant
dose-dependently protected mice from cocaine-induced convul-
sions and lethality. Intraperitoneal administration of cocaine 180
mg/kg produced convulsions and lethality in all tested mice (n
) 6). Pretreatment with the BChE mutant (i.e., 1 min prior to
cocaine administration) dose-dependently protected mice against
cocaine-induced convulsions and lethality. In particular, the
BChE mutant at 0.01 and 0.03 mg produced full protection in
mice from cocaine overdose induced by a lethal dose of cocaine
180 mg/kg (p < 0.05). The in vivo data show that A199S/
F227A/S287G/A328W/Y332G BChE is indeed roughly 10-fold
more potent than CocE for the in vivo protection of mice from
cocaine-induced lethality under the same experimental condition.
The encouraging outcome of this study suggests that the
unique virtual screening approach (depicted in Figure 2) based
on the transition-state simulations and subsequent energetic
calculations is promising for rational enzyme redesign and drug
discovery. The general design approach can be used to rationally
design mutants of any enzyme. When BChE and cocaine in
Figure 2 are replaced by another enzyme and its own substrate,
one still can carry out the similar QM/MM reaction coordinate
calculations on the enzymatic reaction to identify the rate-
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A R T I C L E S
Zheng et al.
ES complex for wild-type BChE with (-)-cocaine in water by using
Amber8 program package.30 Our previous MD simulations12 on
the prereactive BChE-(-)-cocaine complex (ES) started from the
X-ray crystal structure32 deposited in the Protein Data Bank (pdb
code: 1POP). The present MD simulations on the transition state
for the first step (TS1) were performed in such a way that bond
lengths of the transition bonds in the transition state were all
constrained to be the same as those obtained from our previous ab
initio reaction coordinate calculations on the model reaction system
of wild-type BChE.21 A sufficiently long MD simulation with the
transition bonds constrained should lead to a reasonable protein
environment stabilizing the reaction center in the transition-state
structure simulated. Further, the simulated TS1 structure for wild-
type BChE with (-)-cocaine was used to build the initial structures
of TS1 for the examined BChE mutants with (-)-cocaine; only
the side chains of mutated residues needed to be changed.
environment part, by cutting a covalent σ-bond Y-X. Y and X
refer to boundary atoms of the environment part and the active
part, respectively. Instead of using a hydrogen atom to cap the free
valence of X atom as in the conventional link-atom approach, a
pseudobond Yps-X is formed by replacing the Y atom with a one-
free-valence boundary Y atom (Yps). The Yps atom is parametrized
to make the Yps-X pseudobond mimic the original Y-X bond with
similar bond length and strength and also to have similar effects
on the rest of the active part.34,35 In the pseudobond approach, the
Y
ps atom and all atoms in the active part form a well-defined QM
subsystem which can be treated by a QM method. Excluding the
Y atom, the remaining atoms in the environment part form the MM
subsystem treated by a MM method. The pseudobond ab initio QM/
MM approach has been demonstrated to be powerful in studies of
enzyme reactions.36-38
In order to use exactly the same Amber force field in our QM/
MM calculations as that used in our MD simulations, we developed
and used a revised version of Gaussian03 and Amber8 programs,
instead of the revised Gaussian03 and Tinker programs, to perform
the QM/MM calculations in this study. In all of our QM/MM
calculations, all atoms of (-)-cocaine and the side chains of S198,
H438, and E325 were considered as the QM atoms, whereas the
other atoms were regarded as MM atoms. Our QM/MM calculations
were performed at the B3LYP/6-31*:Amber level, i.e., the QM
calculations were carried out at the B3LYP/6-31G* level, whereas
the MM calculations were carried out by using the Amber force
field implemented in the Amber8 program. The geometry optimiza-
tions were converged to the default criteria of the Gaussian03 (for
the QM part) and Amber8 (for the MM part) programs. The QM/
MM-optimized ES, TS1, and INT geometries were used to perform
vibrational frequency calculations on the QM subsystem at the same
QM/MM level. The vibrational frequency calculations confirmed
that the optimized ES and INT1 geometries are indeed associated
with local minima on the potential energy surface, whereas the
optimized TS1 geometry is indeed associated with a first-order
saddle point on the potential energy surface.
The partial atomic charges for the nonstandard residue atoms,
including cocaine atoms, in the TS1 structures were calculated by
using the RESP protocol implemented in the Antechamber module
of the Amber8 package following electrostatic potential (ESP)
calculations at ab initio HF/6-31G* level using Gaussian03 pro-
gram.33 The geometries used in the ESP calculations came from
those obtained from the previous ab initio reaction coordinate
calculations,21 but the functional groups representing the oxyanion
hole were removed. Thus, residues G116, G117, and A199 were
the standard residues as supplied by Amber8 in the MD simulations.
The general procedure for carrying out the MD simulations in water
is essentially the same as that used in our previously reported other
computational studies.12,21,30,32 Each aforementioned starting TS1
structure was neutralized by adding chloride counterions and was
solvated in a rectangular box of TIP3P water molecules with a
minimum solute-wall distance of 10 Å. The total numbers of atoms
in the solvated protein structures for the MD simulations are nearly
70 000, although the total number of atoms of BChE and (-)-
cocaine is only 8417 (for the wild-type BChE). All of the MD
simulations were performed by using the Sander module of Amber8
package. The solvated systems were carefully equilibrated and fully
energy minimized. These systems were gradually heated from T )
10 K to T ) 298.15 K in 30 ps before running the MD simulation
at T ) 298.15 K for 1 ns or longer, making sure that we obtained
a stable MD trajectory for each of the simulated TS1 structures.
The time step used for the MD simulations was 2 fs. Periodic
boundary conditions in the NPT ensemble at T ) 298.15 K with
Berendsen temperature coupling and P ) 1 atm with isotropic
molecule-based scaling were applied. The SHAKE algorithm was
used to fix all covalent bonds containing hydrogen atoms. The
nonbonded pair list was updated every 10 steps. The particle mesh
Ewald (PME) method was used to treat long-range electrostatic
interactions. A residue-based cutoff of 10 Å was utilized to the
noncovalent interactions. The coordinates of the simulated systems
were collected every 1 ps during the production MD stages.
2. QM/MM Calculations. All of the QM/MM calculations were
performed by a pseudobond QM/MM method.34,35 The pseudobond
QM/MM method was initially implemented in revised Gaussian03
and Tinker programs.34,35 The revised Gaussian03 and Tinker
programs can be used to carry out the QM and MM parts of the
QM/MM calculation iteratively until the full self-consistency is
achieved. The pseudobond QM/MM method uses a seven-valence-
electron atom with an effective core potential constructed to replace
the boundary atom of the environment part and to form a
pseudobond with the boundary atom of the active part. The main
idea of the pseudobond approach is as follows: one considers that
a large molecule is partitioned into two parts, an active part and an
Most of the MD simulations and QM/MM calculations were
performed in parallel on an HP Superdome (with 256 processors)
and an IBM X-series cluster (with 1360 processors) at the Center
for Computational Sciences, University of Kentucky. Some com-
putations were carried out on a 34-processors IBM x335 Linux
cluster and SGI Fuel workstations in our own laboratory.
3. Materials for in Vitro Studies. Cloned pfu DNA polymerase
and Dpn I endonuclease were obtained from Stratagene (La Jolla,
CA). [3H](-)-cocaine (50 Ci/mmol) was purchased from Perkin-
Elmer Life Sciences (Boston, MA). The expression plasmid pRc/
CMV was a gift from Dr. O. Lockridge, University of Nebraska
Medical Center (Omaha, NE). All oligonucleotides were synthesized
by the Integrated DNA Technologies, Inc. (Coralville, IA). The
QIAprep spin plasmid miniprep kit and Qiagen plasmid purification
kit and QIAquick PCR purification kit were obtained from Qiagen
(Santa Clarita, CA). Human embryonic kidney 293T/17 cells were
from ATCC (Manassas, VA). Dulbecco’s modified Eagle’s medium
(DMEM) was purchased from Fisher Scientific (Fairlawn, NJ).
3,3′,5,5′-Tetramethylbenzidine (TMB) was obtained from Sigma
(Saint Louis, MO). Antibutyrylcholinesterase (mouse monoclonal
antibody, product no. HAH00201) was purchased from Antibody-
Shop (Gentofte, Denmark), and goat antimouse IgG HRP conjugate
was from Zymed (San Francisco, CA).
4. Site-Directed Mutagenesis, Protein Expression, and in
Vitro Activity Assay. Site-directed mutagenesis of human BChE
cDNA was performed by using the QuikChange method.39 Muta-
tions were generated from wild-type human BChE in a pRc/CMV
(32) Nicolet, Y.; Lockridge, O.; Masson, P.; Fontecilla-Camps, J. C.;
Nachon, F. J. Biol. Chem. 2003, 278, 41141–41147.
(33) Frisch, M. J.; et al. Gaussian 03, revision A.1; Gaussian: Pittsburgh,
PA, 2003.
(34) Zhang, Y.; Lee, T.; Yang, W. J. Chem. Phys. 1999, 110, 46–54.
(35) Zhang, Y. J. Chem. Phys. 2005, 122, 024114.
(36) Corminboeuf, C.; Hu, P.; Tuckerman, M. E.; Zhang, Y. J. Am. Chem.
Soc. 2006, 128, 4530–4531.
(37) Zhang, Y.; Liu, H.; Yang, W. J. Chem. Phys. 2000, 112, 3483–3492.
(38) Hu, P.; Zhang, Y. J. Am. Chem. Soc. 2006, 128, 1272–1278.
(39) Braman, J.; Papworth, C.; Greener, A. Methods Mol. Biol. 1996, 57,
31–44.
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Most Efficient Cocaine Hydrolase
A R T I C L E S
expression plasmid.40 With the use of plasmid DNA as template
and primers with specific base-pair alterations, mutations were made
by polymerase chain reaction with Pfu DNA polymerase, for
replication fidelity. The PCR product was treated with Dpn I
endonuclease to digest the parental DNA template. Modified
plasmid DNA was transformed into Escherichia coli, amplified,
and purified. The DNA sequences of the mutants were confirmed
by DNA sequencing. BChE mutants were expressed in human
embryonic kidney cell line 293T/17. Cells were grown to 80-90%
confluence in 6-well dishes and then transfected by lipofectamine
2000 complexes of 4 µg of plasmid DNA per each well. Cells were
incubated at 37 °C in a CO2 incubator for 24 h, and cells were
moved to 60 mm culture vessel and cultured for 4 more days.
The culture medium (10% fetal bovine serum in DMEM) was
harvested for a BChE activity assay. To measure (-)-cocaine and
benzoic acid, the product of (-)-cocaine hydrolysis catalyzed by
BChE, we used sensitive radiometric assays based on toluene
extraction of [3H]-(-)-cocaine labeled on its benzene ring.41 In
brief, to initiate the enzymatic reaction, 100 nCi of [3H]-(-)-cocaine
was mixed with 100 µL of culture medium. The enzymatic reactions
proceeded at room temperature (25 °C) with varying concentrations
of (-)-cocaine. The reactions were stopped by adding 300 µL of
0.02 M HCl, which neutralized the liberated benzoic acid while
ensuring a positive charge on the residual (-)-cocaine. [3H]benzoic
acid was extracted by 1 mL of toluene and measured by scintillation
counting. Finally, the measured (-)-cocaine concentration-depend-
ent radiometric data were analyzed by using the standard
Michaelis-Menten kinetics so that the catalytic parameters (kcat
and KM) were determined along with the use of an ELISA42
described below.
The ELISA buffers used in the present study are the same as
those described in literature.42 The coating buffer was 0.1 M sodium
carbonate/bicarbonate buffer, pH 9.5. The diluent buffer (EIA
buffer) was potassium phosphate monobasic/potassium phosphate
dibasic buffer, pH 7.5, containing 0.9% sodium chloride and 0.1%
bovine serum albumin. The washing buffer (PBS-T) was 0.01 M
potassium phosphate monobasic/potassium phosphate dibasic buffer,
pH 7.5, containing 0.05% (v/v) Tween-20. All the assays were
performed in triplicate. Each well of an ELISA microtiter plate
was filled with 100 µL of the mixture buffer consisting of 20 µL
of culture medium and 80 µL of coating buffer. The plate was
covered and incubated overnight at 4 °C to allow the antigen to
bind to the plate. The solutions were then removed, and the wells
were washed four times with PBS-T. The washed wells were filled
with 200 µL of diluent buffer and kept shaking for 1.5 h at room
temperature (25 °C). After washing with PBS-T four times, the
wells were filled with 100 µL of antibody (1:8000) and were
incubated for 1.5 h, followed by washing four times. Then, the
wells were filled with 100 µL of goat antimouse IgG HRP conju-
gate complex diluted to a final 1:3000 dilution and were incubated
at room temperature for 1.5 h, followed by washing four times.
The enzyme reactions were started by addition of 100 µL of
substrate (TMB) solution.42 The reactions were stopped after 15
min by the addition of 100 µL of 2 M sulfuric acid, and the
absorbance was read at 460 nm using a Bio-Rad ELISA plate reader.
5. Protein Purification. For the purpose of protein purification,
we also constructed an expression plasmid pSecTag2A/N-His6
encoding the A199S/F227A/S287G/A328W/Y332G mutant of
human BChE for high-level expression. The aforementioned serum
expression system was replaced by Free Style 293 expression
system (Invitrogen). The system used 293F cells to replace the 293T
cells in an orbit shaker bottle instead of using a regular flask. HisPur
cobalt resin (Pierce) was used for the purification leading to highly
purified enzyme (>95% purity). The serum-free medium greatly
facilitated rH-BChE purification processes.
6. In Vivo Studies. 6.1. Subjects and Drugs. Male NIH-Swiss
mice (25-30 g) were obtained from Harlan Sprague-Dawley Inc.
(Indianapolis, IN) and were housed in groups of six mice per cage.
All mice were allowed ad libitum access to food and water and
were maintained on a 12 h light-dark cycle with lights on at 6:30
a.m. in a room kept at a temperature of 21-22 °C. Experiments
were performed in accordance with the Guide for the Care and
Use of Laboratory Animals as adopted and promulgated by the
National Institutes of Health. The experimental protocols were
approved by the University Committee on the Use and Care of
Animals at the University of Michigan.
(-)-Cocaine HCl (National Institute on Drug Abuse, Bethesda,
MD) was dissolved in sterile water and was administered intrap-
eritoneally at a volume of 0.01 mL/g. The BChE mutant (i.e.,
A199S/F227A/S287G/A328W/Y332G BChE) was diluted to dif-
ference concentrations in phosphate-buffered saline and adminis-
tered intravenously at a volume of 0.2 mL/mouse.
6.2. Behavioral Assay. Cocaine-induced toxicity was character-
ized by the occurrence of convulsions and lethality. Cocaine-induced
convulsions were defined as loss of righting posture for at least 5 s
with the simultaneous presence of clonic limb movements.31
Lethality was defined as cessation of observed movement and
respiration. Following cocaine administration, mice were im-
mediately placed individually in Plexiglas containers (16 cm × 28
cm × 20 cm high) for observation. The presence or absence of
convulsions and lethality were recorded for 60 min following
cocaine administration.
6.3. Drug Administration. The mouse was placed in a small
restraint chamber (outer tube diameter, 30 mm; inner tube diameter,
24 mm; model no. BS4-34-0012, Harvard Apparatus, Inc., Holliston,
MA) that left the tail exposed. The tail was cleansed with an alcohol
wipe, and a 30G1/2 precision glide needle (Fisher Scientific,
Pittsburgh, PA) was inserted into one of the side veins for infusion.
The intravenous injection volume of BChE mutant (i.e., 0, 0.01,
and 0.03 mg) was 0.2 mL per mouse, and it was given 1 min before
intraperitoneal administration of cocaine 180 mg/kg. To staunch
the bleeding, sterile gauze and pressure were applied to the injection
site.
6.4. Data Analysis. Data from the behavioral toxicity studies
(i.e., percent of mice showing affected responses) were analyzed
with Chi-square probability test with one tail. Protective effects of
the BChE mutant were compared with those of phosphate-buffered
saline. The criterion for significance was set at p < 0.05.
Acknowledgment. This work was supported by NIH (Grants
DA013930 and DA021416). The authors also acknowledge the
Center for Computational Sciences (CCS) at the University of
Kentucky for supercomputing time on an HP Superdome (with 4
nodes and 256 processors) and an IBM X-series cluster with 1360
processors.
(40) Masson, P.; Xie, W.; Froment, M.-T.; Levitsky, V.; Fortier, P.-L.;
Albaret, C.; Lockridge, O. Biochim. Biophys. Acta 1999, 1433, 281–
293.
Supporting Information Available: Complete refs 30 and 33.
This material is available free of charge via the Internet at http://
pubs.acs.org.
(41) Sun, H.; Shen, M. L.; Pang, Y. P.; Lockridge, O.; Brimijoin, S.
J. Pharmacol. Exp. Ther. 2002, 302, 710–716.
(42) Brock, A.; Mortensen, V.; Loft, A. G. R.; Nørgaard-Pedersen, B.
J. Clin. Chem. Clin. Biochem. 1990, 28, 221–224.
JA803646T
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