An improved cocaine hydrolase: The A328Y mutant of human butyrylcholinesterase is 4-fold more efficient

Article abstract of DOI:10.1124/mol.55.1.83

Butyrylcholinesterase (BChE) has a major role in cocaine detoxication. The rate at which human BChE hydrolyzes cocaine is slow, with ak(cat) of 3.9 min^-1 and K(m) of 14 μM. Our goal was to improve cocaine hydrolase activity by mutating residues near the active site. The mutant A328Y had a k(cat) of 10.2 min^-1 and K(m) of 9 μM for a 4-fold improvement in catalytic efficiency (k(cat)/Km). Since benzoylcholine (k(cat) 15,000 min^{- 1}) and cocaine form the same acyl-enzyme intermediate but are hydrolyzed at 4000-fold different rates, it was concluded that a step leading to formation of the acyl-enzyme intermediate was rate-limiting. BChE purified from plasma of cat, horse, and chicken was tested for cocaine hydrolase activity. Compared with human BChE, horse BChE had a 2-fold higher k(cat) but a lower binding affinity, cat BChE was similar to human, and chicken BChE had only 10% of the catalytic efficiency. Naturally occurring genetic variants of human BChE were tested for cocane hydrolase activity. The J and K variants (E497V and A539T) had k(cat) and K(m) values similar to wild type, but because these variants are reduced to 66 and 33% of normal levelsin human blood respectively, people with these variants may be at risk for cocaine toxicity. The atypical variant (D70G) had a 10 fold lower binding affinity for cocaine, suggesting that persons with the atypical variant of BChE may experience severe or fatal cocaine intoxication when administered a dose of cocaine that is not harmful to others.

Full text of DOI:10.1124/mol.55.1.83

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MOLECULAR PHARMACOLOGY, 55:83–91 (1999).
An Improved Cocaine Hydrolase: The A328Y Mutant of Human
Butyrylcholinesterase is 4-fold More Efficient
WEIHUA XIE, CIBBY VARKEY ALTAMIRANO, CYNTHIA F. BARTELS, ROBERT J. SPEIRS, JOHN R. CASHMAN, and
OKSANA LOCKRIDGE
Eppley Institute and Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska
(W.X., C.V.A., O.L.) and Human BioMolecular Research Institute, Seattle, Washington (R.J.S, J.R.C.)
Received April 1, 1998; accepted September 15, 1998
This paper is available online at http://www.molpharm.org
ABSTRACT
Butyrylcholinesterase (BChE) has a major role in cocaine de- affinity, cat BChE was similar to human, and chicken BChE had
toxication. The rate at which human BChE hydrolyzes cocaine only 10% of the catalytic efficiency. Naturally occurring genetic
is slow, with a kcat of 3.9 minϪ1 and Km of 14 ␮M. Our goal was variants of human BChE were tested for cocaine hydrolase
to improve cocaine hydrolase activity by mutating residues activity. The J and K variants (E497V and A539T) had kcat and
near the active site. The mutant A328Y had a kcat of 10.2 minϪ1 Km values similar to wild-type, but because these variants are
and Km of 9 ␮M for a 4-fold improvement in catalytic efficiency reduced to 66 and 33% of normal levels in human blood,
(kcat/Km). Since benzoylcholine (kcat 15,000 minϪ1) and cocaine respectively, people with these variants may be at risk for
form the same acyl-enzyme intermediate but are hydrolyzed at cocaine toxicity. The atypical variant (D70G) had a 10-fold
4000-fold different rates, it was concluded that a step leading lower binding affinity for cocaine, suggesting that persons with
to formation of the acyl-enzyme intermediate was rate-limiting. the atypical variant of BChE may experience severe or fatal
BChE purified from plasma of cat, horse, and chicken was cocaine intoxication when administered a dose of cocaine that
tested for cocaine hydrolase activity. Compared with human is not harmful to others.
BChE, horse BChE had a 2-fold higher kcat but a lower binding
Cocaine1 abuse is a medical problem in the United States. came from the laboratory of W. Kalow (Stewart et al., 1977,
About 23 million Americans have used cocaine at least once 1979; Inaba et al., 1978). They recognized that only a small
and approximately 5 million are habitual users (Das, 1993).
The number of cocaine-related emergency room visits is
about 100,000 annually (Schrank, 1992). Among a total of
14,843 residents of New York City who received fatal injuries
from 1990 through 1992, 26.7% had cocaine or a metabolite
in their urine or blood (Marzuk et al., 1995). Life-threatening
symptoms due to cocaine toxicity include grand-mal seizures,
cardiac arrest, stroke, and drug-induced psychosis accompa-
nied by elevated body temperature (Rich and Singer, 1991;
Das, 1993; Warner, 1993).
percentage of cocaine appeared unchanged in the urine of
humans and that the major metabolites resulted from the
hydrolytic splitting of ester bonds (Fig. 1). Human plasma
was known to contain only two major esterases, BChE and
paraoxonase. The esterase in blood that hydrolyzes cocaine
was identified as BChE by showing that diisopropyl fluoro-
phosphate, eserine, and sodium fluoride, three characteristic
inhibitors of BChE, inhibited hydrolysis of cocaine. By con-
trast, EDTA, an inhibitor of paraoxonase, did not inhibit
hydrolysis of cocaine by plasma. Furthermore, purified BChE
accounted for all the cocaine hydrolase activity seen in
plasma. The metabolite produced by BChE in the test tube
was ecgonine methyl ester (Fig. 1); ecgonine methyl ester was
also a major metabolite found in urine, thus supporting a role
for BChE in metabolism of cocaine (Inaba et al., 1978). A
second metabolite found in high amounts in urine was ben-
zoylecgonine (Fig. 1). This metabolite is produced by sponta-
neous hydrolysis at alkaline pH as well as by liver carboxy-
lesterase (Brzezinski et al., 1994).
There is evidence that butyrylcholinesterase (BChE, EC
3.1.1.8) is the major detoxicating enzyme of cocaine. The first
experiments that identified BChE as a cocaine hydrolase
Supported by the Nebraska Affiliate of the American Heart Association
Grant 9707841S (to O.L.), AASERT Award DAAG55-07-1-0244 from the U.S.
Army Research Office (to O.L.), Minority Student and Sciences Teacher Train-
ing Program Grant R25RR10280 from the National Center for Research Re-
sources, National Cancer Institute Grant P30 CA36727 to the Eppley Insti-
tute, National Institutes of Health Grants DA08531 and DA00269 (to J.R.C.)
and DA011707 (to O.L.), and by U.S. Army Medical Research and Materiel
Command Grant DAMD17-97-1-7349 (to O.L.). The opinions or assertions
contained herein belong to the authors and should not be construed as the
official views of the U.S. Army or the Department of Defense.
Animal studies showed that administration of purified hu-
man BChE protected mice and rats from the toxic effects of
cocaine. Hoffman et al. (1996) gave mice an i.p. injection of
1 Cocaine possesses the “(Ϫ)” configuration unless otherwise indicated.
ABBREVIATIONS: BChE, butyrylcholinesterase enzyme.
83

84
Xie et al.
purified human BChE and, 1 h later, an injection of cocaine. oligonucleotides were synthesized by the Molecular Biology Core
Facility at the University of Nebraska Medical Center. Procain-
amide-Sepharose was a gift from Dr. B. P. Doctor, Walter Reed Army
Research Institute (Washington, DC). Other reagents were DE52
from Whatman (Maidstone, England); echothiophate iodide from
Wyeth-Ayerst (Rouses Point, NY); and butyrylthiocholine iodide,
benzoylcholine chloride, diisopropyl fluorophosphate, and methio-
nine sulfoximine from Sigma Chemical Co. (St. Louis, MO). (Ϫ)-
Cocaine hydrochloride was purchased from Sigma after obtaining a
license from the Department of Justice, Drug Enforcement Agency
(Washington, DC). (ϩ)-Cocaine was provided by the National Insti-
tute on Drug Abuse Research Resources Drug Supply System (Rock-
ville, MD). Cat plasma and chicken serum were from Pel-Freez
Biologicals (Rogers, AK). Horse serum was from Gibco/BRL (Gaith-
ersburg, MD). Highly purified human BChE, 280 U/ml when assayed
with butyrylthiocholine, was a gift from Dr. G. Ghenbot (Centeon
L.L.C., Kankakee, IL).
Mutagenesis and Expression of Recombinant BChE. Muta-
tions in human BChE were made by PCR with Pfu polymerase. Pfu
polymerase was superior to Taq polymerase and DeepVent because
it did not introduce unwanted mutations. The 1.8-kb fragment con-
taining the mutation was cloned into the plasmid pGS and com-
pletely resequenced to make certain that only the desired mutation
was present. The plasmid pGS is identical with pRc/CMV (Invitro-
gen, Carlsbad, CA) except that the Neo gene of pRc/CMV has been
replaced by rat glutamine synthetase. Transfection of CHO-KI cells
by calcium phosphate coprecipitation was followed by selection of
colonies in glutamine-free, serum-free medium Ultraculture contain-
ing 50 ␮M methionine sulfoximine. Colonies expressing the highest
levels of BChE activity were expanded. For collection of large vol-
umes of secreted BChE, cells in 1-liter roller bottles were fed every 2
or 3 days with 100 ml of Ultraculture containing 25 ␮M methionine
sulfoximine followed by 100 ml of Dulbecco’s modified Eagle’s medi-
um/Ham’s F12 without glutamine. Medium was collected from the
same roller bottle for 3 to 6 months.
Purification of BChE from Culture Medium. BChE was pu-
rified from 6 to 12 liters of culture medium containing secreted
BChE. Culture medium was reduced in volume to 800 ml by pouring
the liquid into a 5-cm (flat diameter) dialysis bag and packing table
sugar around the bag. The top of the bag was tied with a rubber band
to allow refilling. The 800-ml viscous liquid was diluted with 4
volumes of 20 mM potassium phosphate 1 mM EDTA, pH 7, to
reduce the salt concentration to allow the BChE to stick to the
affinity gel. Particulate matter was removed by centrifugation.
BChE activity was purified on procainamide-Sepharose eluted with
0.2 M procainamide, followed by ion exchange chromatography on
DE52 and elution with a gradient of sodium chloride (Lockridge,
1990). The BChE was dialyzed in an Amicon Stirred cell with a PM10
membrane to concentrate the enzyme and to adjust the buffer to 0.1
M potassium phosphate, 1 mM EDTA, pH 7. Preparations used for
(Ϫ)-cocaine hydrolysis had activities with 1 mM butyrylthiocholine of
20 to 150 units/ml where unit is defined as ␮moles hydrolyzed per
minute.
The cocaine dose of 150 mg/kg i.p. caused seizures and death
in 100% of animals when animals were not pretreated with
BChE. By contrast, pretreatment with BChE protected 70%
of mice from death and 60% of mice from seizures. Similarly,
Lynch et al. (1997) pretreated rats with BChE and found that
rats were protected from the lethal effects of cocaine as well
as from hypertension and arrhythmia. BChE was also effec-
tive when it was given to rats after cocaine. Rats had convul-
sions and died within 9 min of receiving 80 mg/kg cocaine i.p.,
but were protected when they were injected with BChE 3 min
after receiving cocaine. It was concluded that BChE could be
an effective therapy for the treatment of life-threatening
cocaine-induced toxicity (Mattes et al., 1997).
Although BChE protects against cocaine toxicity by inac-
tivating cocaine, it acts slowly. Our goal was to increase the
catalytic efficiency of BChE toward (Ϫ)-cocaine by increasing
its binding affinity and hydrolysis rate. Of the 24 mutants of
human BChE tested, only the A328Y mutant had an im-
proved catalytic efficiency for cocaine hydrolysis. In addition,
we tested purified BChE from cat, chicken, and horse and
found that the catalytic efficiency (kcat/Km) of BChE from all
three animals was lower than that of wild-type human
BChE, despite a 2-fold higher kcat in horse BChE.
Another area we investigated was the catalytic efficiency of
some of the naturally occurring genetic variants of BChE
toward cocaine, including the atypical variant, the K variant,
and the J variant. It is established that people with genetic
variants of BChE, particularly the atypical variant (D70G),
are more sensitive to the muscle relaxants succinylcholine
and mivacurium and that this abnormal response is due to a
decreased binding affinity for these drugs (Lockridge, 1990;
Kalow and Grant, 1995). Our results suggest that people
with the atypical variant of BChE have an increased risk of
suffering complications from cocaine use.
Materials and Methods
Materials. Reagents for site-directed mutagenesis and expression
included Pfu polymerase (Stratagene, La Jolla, CA), the expression
plasmid pGS (gift from Dr. Tyler White, Scios Nova Inc., Mountain
View, CA), Ultraculture without L-glutamine (BioWhittaker, Fisher
Scientific Co., Fairlawn, NJ), Dulbecco’s modified Eagle’s medium
and Ham’s F12, 50/50 mix without L-glutamine (Mediatech Cellgro,
Fisher Scientific Co.), CHO-KI cells (No. CCL 61; American Type
Culture Collection, Rockville, MD), Qiagen plasmid purification kit
and QIAquick PCR purification kit (Qiagen, Santa Clarita, CA). All
Purification of BChE from Animal Sera. BChE was purified
from 3 liters of chicken serum, 1.5 liters of horse serum, and 0.35
liter of cat plasma by ammonium sulfate precipitation, ion exchange
chromatography, and affinity chromatography on procainamide-
Sepharose (Main et al., 1974; Ralston et al., 1983). The preparations
used for (Ϫ)-cocaine hydrolysis had activities with 1 mM butyrylthio-
choline of 49 U/ml (chicken BChE), 92 U/ml (horse BChE), and 9
U/ml (cat BChE).
Titration of Active Sites. The number of active sites per ml of
purified BChE was titrated with echothiophate iodide and with
diisopropyl fluorophosphate. The catalytic rate constant, kcat, was
calculated by dividing Vmax by the concentration of active sites.
Benzoylcholine Hydrolysis. Benzoylcholine chloride concentra-
tion was verified by absorbance at 240 nm where a 0.2 mM stock
solution had an absorbance of 1.874. Km values were determined for
Fig. 1. Metabolism of (Ϫ)-cocaine. The naturally occurring (Ϫ)-cocaine is
the pharmacologically active enantiomer.

Cocaine Hydrolase Activity of Butyrylcholinesterase
85
the range 12.5 to 50 ␮M benzoylcholine in 0.1 M potassium phos- 1993; Masson et al., 1997). The equation for substrate activation
phate, pH 7.0, at 25°C. Hydrolysis of benzoylcholine was recorded at (Radic et al., 1993) was used to calculate the kinetic constants for
240 nm. Hydrolytic activity was calculated from the difference in butyrylthiocholine hydrolysis with the Sigma Plot Computer Pro-
molar absorptivity of benzoylcholine and benzoic acid, ⌬E ϭ 6700 gram. The Km value describes binding at low substrate concentra-
MϪ1 cmϪ1
.
tions, 0.01 to 0.1 mM; the Kss value describes binding at high sub-
Cocaine Hydrolysis. (Ϫ)-Cocaine and (ϩ)-cocaine stock solutions strate concentrations, 0.4 to 40 mM; the b value is the ratio of Vmax
of 0.1 M were made in water and frozen. Cocaine was stable when at high substrate concentration divided by Vmax at low substrate
frozen in water, but unstable in phosphate buffer at pH 7; stability concentration. When the b value is greater than one there is sub-
was checked by high-performance liquid chromatography (HPLC). strate activation; when the b value is less than one there is substrate
Enzyme-catalyzed hydrolysis of cocaine was recorded on a tempera- inhibition; when the b value is equal to one there is neither substrate
ture-equilibrated Gilford Spectrophotometer at 240 nm where the activation nor substrate inhibition and double reciprocal plots are
difference in molar absorptivity between substrate and product was linear.
⌬E ϭ 6,700 MϪ1 cmϪ1 (Gatley, 1991). Km values were determined in
HPLC. A Waters 625 LC System with Waters 486 Tunable Ab-
0.1 M potassium phosphate pH 7.0 at 30°C for (Ϫ)-cocaine and at sorbance Detector and Waters Delta Pak C18, 300-Å, 5-␮ column
25°C for (ϩ)-cocaine. Vmax and Km values were calculated using were used. The elution buffer was an 80:20 mixture of 0.05 M
Sigma Plot for Macintosh computer (Jandel Scientific).
potassium phosphate, pH 3.0, and acetonitrile. The flow rate was 0.5
Butyrylthiocholine Hydrolysis. Butyrylthiocholine stock solu- ml/min. Absorbance was recorded at 220 nm. 100 ␮M (Ϫ)-cocaine
tions of 0.2 M were prepared in water and frozen. Hydrolysis of freshly diluted in 0.1 M potassium phosphate, pH 7.0, was incubated
butyrylthiocholine was recorded at 412 nm in the presence of 0.5 mM at 37°C in the presence of 0.2 ␮M wild-type BChE or the A328Y
dithiobisnitrobenzoic acid in 0.1 M potassium phosphate, pH 7.0, at mutant of BChE. 100-␮l aliquots were removed for analysis of hy-
25°C. Activity was calculated from the molar extinction coefficient of drolysis products by HPLC. The 100-␮l aliquot was acidified to pH 3
13,600 MϪ1 cmϪ1 (Ellman et al., 1961). Units of activity are ex- by the addition of 1 ␮l of 85% phosphoric acid. It was important to
pressed as micromoles substrate hydrolyzed per minute.
acidify the samples to get a single, sharp peak for benzoic acid.
The competitive inhibition constant, Ki, was determined by mea- Protein-containing aliquots were filtered through Millipore Ultra-
suring hydrolysis of butyrylthiocholine (12.5–100 ␮M) in the pres- free-MC 10,000 NMWL filter units by centrifugation in a microcen-
ence of (Ϫ)-cocaine (0–100 ␮M) and plotting 1/v versus inhibitor trifuge to remove particulates before injecting the sample on the
concentration in a Dixon plot. Each analysis was repeated three HPLC.
times. The butyrylthiocholine concentrations were below the range
Docking. Cocaine was docked into the active site of BChE with
where substrate activation occurred; plots of 1/v versus 1/S were the FlexiDock program in Sybyl 6.4 on a Silicon Graphics Octane
linear for the range used. For the D70G mutant the butyrylthiocho- computer. The structures of (Ϫ)-cocaine and (ϩ)-cocaine were re-
line concentrations were 105 to 630 ␮M and the (Ϫ)-cocaine concen- trieved from the Cambridge Structural Database where its code
trations were 117 to 702 ␮M.
names are COCAIN10 and COCHCL. The HCl molecule was deleted
Substrate Activation. Lineweaver-Burk plots for butyrylthio- from COCHCL, so that all computations were done with the base
choline hydrolysis are not linear for the range 0.01 to 40 mM bu- form of cocaine. Before the FlexiDock program was run, cocaine was
tyrylthiocholine when the assays are performed in phosphate buffer. manually aligned with butyrylcholine in the model of human BChE
The curvature in this plot has been attributed to substrate activation (Harel et al., 1992), so that the tropane ring of cocaine faced Trp-82,
at concentrations above 0.4 mM butyrylthiocholine (Radic et al., the carboxyl of the benzoic acid ester of cocaine was within 1.5 Å of
TABLE 1
Kinetic constants for recombinant human BChE, determined in 0.1 M potassium phosphate, pH 7.0, at 25°C for butyrylthiocholine and
benzoylcholine and at 30°C for (Ϫ)-cocaine
Butyrylthiocholine
Human BChE
Km
Kss
b
kcat
bkcat
␮M
mM
minϪ1
minϪ1
Wild-type
N68Y/Q119Y/A277W
Q119Y
23 Ϯ 2
59 Ϯ 10
11 Ϯ 1
26 Ϯ 8
83 Ϯ 4
9 Ϯ 1
20 Ϯ 1
15 Ϯ 1
24 Ϯ 4
27 Ϯ 3
1.4 Ϯ 0.2
3.4 Ϯ 0.3
1.6 Ϯ 0.6
0.5 Ϯ 0.1
21 Ϯ 12
0.3 Ϯ 0.1
0.7 Ϯ 0.3
0.2 Ϯ 0.2
1.3 Ϯ 0.1
2.0 Ϯ 0.4
2.5 Ϯ 0.1
4.7 Ϯ 0.4
10.9 Ϯ 2.7
2.5 Ϯ 0.5
1.6 Ϯ 0.2
3.2 Ϯ 1.1
1.2 Ϯ 0.2
1.2 Ϯ 0.2
3.3 Ϯ 0.2
2.4 Ϯ 0.1
33,900 Ϯ 1,800
11,700 Ϯ 900
6,300 Ϯ 1,700
19,700 Ϯ 800
11,500 Ϯ 200
14,100 Ϯ 4,900
32,400 Ϯ 1,000
38,800 Ϯ 8,400
21,200 Ϯ 1,300
19,000 Ϯ 1,000
84,900 Ϯ 4,600
55,000 Ϯ 4,500
69,000 Ϯ 18,100
49,200 Ϯ 2,100
18,500 Ϯ 300
45,200 Ϯ 15,700
38,900 Ϯ 1,100
48,500 Ϯ 9,900
69,900 Ϯ 4,400
45,500 Ϯ 2,500
Q119Y/V288F/A328Y
E197Q
V288F
A328F
A328Y
F329A
F329S
Benzoylcholine
(Ϫ)-Cocaine
Km
kcat
Km
kcat
kcat/Km
␮M
minϪ1
␮M
minϪ1
MϪ1 minϪ1
Wild-type
N68Y/Q119Y/A277W
Q119Y
8 Ϯ 1
20 Ϯ 1
31 Ϯ 1
23 Ϯ 2
29 Ϯ 2
15 Ϯ 1
8 Ϯ 2
12 Ϯ 1
10 Ϯ 1
N/A
14,500 Ϯ 200
5,500 Ϯ 100
10,600 Ϯ 200
6,600 Ϯ 200
7,200 Ϯ 200
11,700 Ϯ 300
11,800 Ϯ 300
13,300 Ϯ 400
10,100 Ϯ 200
N/A
14 Ϯ 1
60 Ϯ 12
56 Ϯ 10
33 Ϯ 6
17 Ϯ 1
17 Ϯ 2
24 Ϯ 4
9 Ϯ 1
3.9 Ϯ 0.1
1.7 Ϯ 0.2
2.0 Ϯ 0.2
2.3 Ϯ 0.2
0.1 Ϯ 0.01
1.0 Ϯ 0.1
5.8 Ϯ 0.4
10.2 Ϯ 0.4
2.7 Ϯ 1.0
1.9 Ϯ 0.3
280,000
28,000
36,000
70,000
6,000
60,000
240,000
1,130,000
21,000
46,000
Q119Y/V288F/A328Y
E197Q
V288F
A328F
A328Y
F329A
128 Ϯ 20
41 Ϯ 18
F329S

86
Xie et al.
Ser-198, and the benzene ring of cocaine was in the acyl binding was to allow (Ϫ)-cocaine to fit more easily into the active site.
pocket of BChE. In FlexiDock the binding pocket was defined as all
amino acids within 4 Å of butyrylcholine. After the binding pocket
was defined, the butyrylcholine molecule was extracted. All atoms in
the binding pocket, except atoms in rings and double-bonded atoms,
were defined as rotatable, thus yielding 124 rotatable bonds in BChE
and 7 rotatable bonds in cocaine.
The active site gorge has several named regions including the
active site Ser-198 at the bottom of the gorge, the acyl bind-
ing pocket formed by Leu-286 and Val-288 (Harel et al.,
1992), the cation ␲ site Trp-82, and the peripheral anionic
site at the mouth of the gorge defined by Asp-70 (Masson et
al., 1997).
(؊)-Cocaine Hydrolase Activity. Each of the human
BChE mutants in Table 1 was tested for activity with three
substrates: butyrylthiocholine, benzoylcholine and (Ϫ)-co-
caine. The maximum rate of hydrolysis of butyrylthiocholine
at high substrate concentration (bkcat) was higher for wild-
type BChE than for the mutants. Km values for butyrylthio-
choline hydrolysis were lower for three mutants Q119Y,
V288F, and A328Y. Since benzoylcholine and cocaine have
benzoate as a common structural feature, it was of interest to
see whether kinetic constants for benzoylcholine correlated
with those for cocaine. Table 1 shows that Km values for
wild-type BChE and for all mutants were in the micromolar
range for both benzoate-containing substrates. In general,
the Km values for cocaine were higher than for benzoylcho-
line, although we expect that this is due to the higher tem-
perature at which cocaine hydrolysis was measured. Two
mutants, E197Q and A328Y, had a lower Km for cocaine than
for benzoylcholine. Benzoylcholine was hydrolyzed at the
fastest rate (highest kcat) by wild-type BChE. By contrast,
(Ϫ)-cocaine was hydrolyzed faster by the A328Y mutant (kcat
Results
Choice of Mutations to Make. Amino acids in the lining
of the active site gorge were selected for mutation. The goal
TABLE 2
Binding constant of (Ϫ)-cocaine to mutants of BChE. Ki values were
determined by measuring inhibition of butyrylthiocholine hydrolysis by
cocaine in 50 mM potassium phosphate, pH 7.4, at 25°C
BChE
Ki
␮M
Wild-type
D70G
D70N
11 Ϯ 1
201 Ϯ 32
486 Ϯ 31
440 Ϯ 25
302 Ϯ 33
34 Ϯ 35
40 Ϯ 4
G117H
G117K
Q119H
E197D
E197G
L286H
V288H
A328F
A328G
A328H
Y332F
37 Ϯ 2
24 Ϯ 2
55 Ϯ 4
21 Ϯ 1
18 Ϯ 1
ϭ 10.2 minϪ1) and by the A328F mutant (kcat ϭ 5.8 minϪ1
)
27 Ϯ 3
than by wild-type BChE (kcat ϭ 3.9 minϪ1). The data in Table
1 show that neither benzoylcholine nor butyrylthiocholine is
useful for predicting which mutant will have the best activity
with (Ϫ)-cocaine.
22 Ϯ 2
TABLE 3
Comparison of Km and Ki values for (Ϫ)-cocaine. Ki values were
determined by measuring inhibition of butyrylthiocholine hydrolysis by
cocaine in 0.1 M potassium phosphate, pH 7.0, at 30°C
The catalytic efficiency of an enzyme can be measured by
determining kcat/Km. A comparison of kcat/Km values in Table
1 shows that the A328Y mutant was 4-fold more efficient
than wild-type BChE at hydrolyzing (Ϫ)-cocaine. No other
mutant outperformed wild-type BChE.
(Ϫ)-Cocaine
Human BChE
Km
Ki
␮M
(؉)-Cocaine Hydrolase Activity. (ϩ)-Cocaine is the un-
natural isomer and has no pharmacologic activity. Gatley
(1991) reported that (ϩ)-cocaine was hydrolyzed orders of
magnitude more rapidly than (Ϫ)-cocaine by human wild-
type BChE and that the rate of hydrolysis of (ϩ)-cocaine was
similar to that of benzoylcholine. We confirmed this result for
Wild-type
14 Ϯ 1
172 Ϯ 36
24 Ϯ 4
9 Ϯ 2
12 Ϯ 2
10 Ϯ 2
13 Ϯ 3
14 Ϯ 2
212 Ϯ 18
10 Ϯ 2
8 Ϯ 2
11 Ϯ 2
9 Ϯ 2
8 Ϯ 2
Atypical (D70G)
A328F
A328Y
J variant (E497V)
J/K variant (E497V/A539T)
K variant (A539T)
TABLE 4
Kinetic constants for native BChE purified from animal plasma determined in 0.1 M potassium phosphate, pH 7.0, at 25°C for butyrylthiocholine
and benzoylcholine, and at 30°C for (Ϫ)-cocaine
Butyrylthiocholine
Native BChE
Km
Kss
b
kcat
bkcat
␮M
mM
minϪ1
minϪ1
Cat
44 Ϯ 2
12 Ϯ 1
23 Ϯ 2
60 Ϯ 8
3.0 Ϯ 0.3
13.0 Ϯ 2.0
1.4 Ϯ 0.2
1.0 Ϯ 0.2
2.1 Ϯ 0.1
2.4 Ϯ 0.1
2.5 Ϯ 0.1
2.1 Ϯ 0.2
105,100 Ϯ 2,500
11,400 Ϯ 100
33,900 Ϯ 1,800
66,600 Ϯ 600
220,600 Ϯ 5,300
27,300 Ϯ 300
84,900 Ϯ 4,600
139,900 Ϯ 12,600
Chicken
Human
Horse
Benzoylcholine
(Ϫ)-Cocaine
Km
kcat
Km
kcat
kcat/Km
␮M
minϪ1
␮M
minϪ1
MϪ1 minϪ1
Cat
8 Ϯ 1
8 Ϯ 1
12,900 Ϯ 100
690 Ϯ 9
14,500 Ϯ 200
11,800 Ϯ 400
10 Ϯ 3
67 Ϯ 19
14 Ϯ 1
45 Ϯ 5
2.7 Ϯ 0.1
1.4 Ϯ 0.2
3.9 Ϯ 0.1
7.5 Ϯ 0.3
270,000
21,000
280,000
170,000
Chicken
Human
Horse
8 Ϯ 1
21 Ϯ 2

Cocaine Hydrolase Activity of Butyrylcholinesterase
87
human wild-type BChE. Our values for (ϩ)-cocaine were Km A277L and P285L at the mouth of the gorge, and F398I at the
ϭ 10 ␮M, kcat ϭ 7500 minϪ1 and the values for benzoylcho- bottom of the gorge. Table 4 shows that cat BChE hydrolyzed
line were Km ϭ 8 ␮M, kcat ϭ 15,000 minϪ1. We also measured
butyrylthiocholine about 3 times faster compared with wild-
hydrolysis of (ϩ)-cocaine by the A328Y mutant. The kinetic
constants at 25°C in 0.1 M potassium phosphate, pH 7.0,
were Km ϭ 7 ␮M, kcat ϭ 6,000 minϪ1. We conclude that the
A328Y mutation did not significantly alter catalytic effi-
ciency toward (ϩ)-cocaine.
type human BChE, but it hydrolyzed benzoylcholine and
(Ϫ)-cocaine somewhat more slowly. Cat BChE has Km values
that are similar to those of human BChE for the three sub-
strates in Table 4. We concluded that cat BChE was excep-
tionally active toward butyrylthiocholine, but no better than
human BChE for hydrolysis of (Ϫ)-cocaine.
Horse BChE, like cat BChE, has three amino acid differ-
ences in the active site gorge compared with human BChE
(B. P. Doctor, personal communication). They are A277V and
P285L at the mouth of the gorge, and F398I at the bottom of
the gorge. Horse BChE had a 2-fold higher kcat for cocaine
than human BChE, but its binding affinity was decreased so
that its catalytic efficiency for hydrolysis of cocaine was not
improved relative to human BChE.
The amino acid sequence of chicken BChE is only partially
known (Jean Massoulie´, personal communication). Chicken
BChE has 10 amino acid differences in the active site gorge:
N68L, S79T, G117S, Q119E, A277V, T284S, P285L, S287H,
V288I, and A328S. Purified chicken BChE had 34% of the
specific activity of human BChE with butyrylthiocholine, 5%
with benzoylcholine, and 36% with cocaine (Table 4). The
Inhibition Constants. Because (Ϫ)-cocaine is hydrolyzed
20,000-fold more slowly than butyrylthiocholine and 4,000-
fold more slowly than benzoylcholine, the amount of BChE
required to measure (Ϫ)-cocaine hydrolysis is relatively high.
High concentrations of mutant BChE were not available for
all mutants we wanted to test. Therefore mutants in Table 2
were analyzed for cocaine binding affinity by measuring Ki
values rather than Km values. None of the mutants in Table
2 had a higher binding affinity for (Ϫ)-cocaine (that is, a
lower Ki value) than wild-type BChE. The validity of Ki
values as a reflection of Km values is demonstrated in Table
3 where Ki and Km values were found to be similar when both
values were measured for the same enzyme preparation un-
der the same conditions. Inhibition was competitive.
Animal BChE. To examine the influence of multiple
amino acid differences on binding affinity and hydrolysis rate
we tested purified BChE from cat, chicken, and horse. The
amino acid sequence of cat BChE (Genbank accession num-
ber AF053483) is 88% identical with human BChE. Of the 70
amino acids that differ, 50 are located on the surface, 13 are
buried, 4 are in the C-terminal region missing from the X-ray
structure, and 3 are in the active site gorge. The three amino
acid differences in the active site gorge of cat BChE are
Fig. 2. Substrate inhibition. A, substrate inhibition by (Ϫ)-cocaine. B,
substrate inhibition by benzoylcholine. Specific activity is expressed as
micromoles hydrolyzed per minute per milligram of BChE protein. Hu-
man BChE is inhibited by (Ϫ)-cocaine concentrations greater than 120
␮M and by benzoylcholine concentrations greater than 50 ␮M.
Fig. 4. HPLC analysis of the products of (Ϫ)-cocaine hydrolysis. Hydro-
lysis of 100 ␮M (Ϫ)-cocaine in 0.1 M potassium phosphate, pH 7.0, 37°C,
was initiated by addition of wild-type BChE or the A328Y mutant of
BChE. Aliquots of 100 ␮l were removed after the indicated minutes of
reaction, acidified to pH 3, filtered, and injected. Absorbance was moni-
tored at 220 nm during isocratic elution with an 80:20 mixture of 0.05 M
potassium phosphate, pH 3.0, and acetonitrile. The product of cocaine
hydrolysis by wild-type and A328Y was benzoic acid, eluting at 10.2 min.
Neither enzyme produced benzoylecgonine. The peak at 3.4 min is a
contaminant in the enzyme storage buffer. All of the (Ϫ)-cocaine was
hydrolyzed within 150 min by 0.2 ␮M wild-type BChE and within 60 min
by 0.2 ␮M A328Y.
Fig. 3. Schematic diagram of the steps in substrate hydrolysis. EOH is
free enzyme; R is either the choline of benzoylcholine or the ecgonine
methyl group of cocaine; Kd is the dissociation constant for the Michaelis-
Menten complex; k2 is the rate constant for formation of the acyl-enzyme
intermediate; k3 is the rate constant for hydrolysis of the acyl-enzyme
intermediate.

88
Xie et al.
second order rate constant, kcat/Km, for hydrolysis of cocaine benzoic acid. Both wild-type BChE and the A328Y mutant
by chicken BChE was 10% of the value for human BChE.
produced benzoic acid, but no benzoylecgonine. This result is
Substrate Inhibition. The first studies on cocaine metab- consistent with the scheme in Fig. 1, where BChE hydrolyzes
olism reported an absence of cocaine hydrolases in human (Ϫ)-cocaine to ecgonine methyl ester and benzoic acid. We
conclude that the methyl ester group of (Ϫ)-cocaine was not
hydrolyzed by the A328Y mutant or by wild-type BChE.
About 1% of the cocaine spontaneously degraded to benzo-
ylecgonine after 150 min in 0.1 M phosphate buffer pH 7.0 at
37°C in the control incubation but not in the enzyme-contain-
ing reactions. Another result from Fig. 4 is confirmation of
the faster catalytic rate of the A328Y mutant. 100 ␮M (Ϫ)-
cocaine was consumed in 140 min by 0.2 ␮M wild-type BChE
and in 52 min by 0.2 ␮M A328Y, a result consistent with a
2.6-fold higher kcat value for the A328Y mutant.
Naturally Occurring Genetic Variants of Human
BChE. Since BChE has a major role in detoxication of co-
caine, it has been suggested that people who are unusually
susceptible to the toxic effects of cocaine are those who carry
a genetic variant of BChE (Kalow and Grant, 1995). We
investigated the atypical variant containing the D70G muta-
tion (McGuire et al., 1989), the J variant containing the
E497V mutation (Bartels et al., 1992a), the K variant con-
taining the A539T mutation (Bartels et al., 1992b) and the
J/K variant containing the double mutation E497V/A539T.
Table 5 shows that the atypical variant has a 10-fold higher
Km value for (Ϫ)-cocaine and a 10-fold lower catalytic effi-
ciency. The J, K and J/K variants have normal binding affin-
ities and normal kcat values for (Ϫ)-cocaine.
blood because the early assays used mM concentrations of
cocaine. Stewart et al. (1977) found that the cocaine concen-
tration had to be reduced to the ␮M range before hydrolysis
could be observed. We examined substrate inhibition with
human, cat, chicken, and horse BChE. Figure 2A shows that
hydrolysis of (Ϫ)-cocaine by human BChE was inhibited
when cocaine concentrations were greater than 120 ␮M. Cat
BChE was inhibited by concentrations greater than 100 ␮M
and chicken BChE by concentrations greater than 140 ␮M.
Horse BChE was not inhibited by cocaine concentrations up
to 170 ␮M. Figure 2A also shows that purified horse BChE
hydrolyzed cocaine more rapidly than purified human BChE,
followed by cat BChE, and last by chicken BChE.
Benzoylcholine and cocaine are the only substrates of
BChE that exhibit substrate inhibition at concentrations in
the ␮M range. In Fig. 2B we examined the pattern of sub-
strate inhibition with benzoylcholine and found that it was
similar but not identical with the pattern with (Ϫ)-cocaine.
Inhibition by benzoylcholine occurred when the benzoylcho-
line concentration was greater than 50 ␮M for human, 100
␮M for cat, 80 ␮M for horse, and 80 ␮M for chicken BChE.
The highest specific activity with benzoylcholine was
achieved by human BChE, followed by cat, horse and
chicken. Chicken BChE was relatively poor at hydrolyzing
both benzoylcholine and (Ϫ)-cocaine.
Rate Limiting Step for Cocaine Hydrolysis. Benzoyl-
choline and cocaine form the same acyl-enzyme intermediate,
in which benzoic acid is esterified to the hydroxyl group of
Ser-198. The fact that (Ϫ)-cocaine is hydrolyzed 4000-fold
more slowly than benzoylcholine by human BChE suggests
that the rate limiting step for hydrolysis of cocaine is a step
leading to the formation of the acyl-enzyme intermediate,
that is, k2 in Fig. 3.
Discussion
A328Y is a Better Cocaine Hydrolase. To explain why
the A328Y mutant hydrolyzes (Ϫ)-cocaine more rapidly than
wild-type BChE, we docked (Ϫ)-cocaine into the active site of
BChE (Fig. 5). Cocaine was positioned with the tropane ring
facing Trp-82 and the benzene ring facing the acyl binding
pocket of BChE. To avoid obstructing the view all residues
Products of (؊)-Cocaine Hydrolysis. To test the possi- were deleted except 328 and 438. Figure 5A shows that in
bility that the A328Y mutant might be hydrolyzing the wild-type BChE the methyl ester group of (Ϫ)-cocaine is very
methyl ester group of (Ϫ)-cocaine, we identified the products close to His-438, overlapping the van der Waals surface of
of hydrolysis by HPLC. Figure 4 shows that the product was His-438, and might affect the function of His-438 in catalysis
TABLE 5
Kinetic constants for naturally occurring genetic variants of human BChE, determined in 0.1 M potassium phosphate, pH 7.0, at 25°C for
butyrylthiocholine and benzoylcholine, and at 30°C for (Ϫ)-cocaine
Butyrylthiocholine
Human BChE
Km
Kss
b
kcat
bkcat
␮M
mM
minϪ1
minϪ1
Wild-type
23 Ϯ 2
150 Ϯ 30
17 Ϯ 2
1.4 Ϯ 0.2
1.2 Ϯ 0.2
1.0 Ϯ 0.1
1.0 Ϯ 0.1
0.7 Ϯ 0.1
2.5 Ϯ 0.1
1.2 Ϯ 0.2
2.9 Ϯ 0.2
2.9 Ϯ 0.1
2.9 Ϯ 0.2
33,900 Ϯ 1,800
32,100 Ϯ 200
32,200 Ϯ 1,900
30,500 Ϯ 1,500
32,500 Ϯ 2,200
84,900 Ϯ 4,600
38,500 Ϯ 300
93,500 Ϯ 5,500
88,400 Ϯ 4,200
94,300 Ϯ 6,500
Atypical variant (D70G)
J variant (E497V)
J/K variant (E497V/A539T)
K variant (A539T)
17 Ϯ 2
17 Ϯ 3
Benzoylcholine
(Ϫ)-Cocaine
Km
kcat
Km
kcat
kcat/Km
␮M
minϪ1
␮M
minϪ1
MϪ1 minϪ1
Wild-type
8 Ϯ 1
40 Ϯ 2
11 Ϯ 1
9 Ϯ 1
14,500 Ϯ 200
15,400 Ϯ 200
18,200 Ϯ 600
13,400 Ϯ 200
15,900 Ϯ 400
14 Ϯ 1
172 Ϯ 36
12 Ϯ 2
3.9 Ϯ 0.1
4.0 Ϯ 0.6
3.7 Ϯ 0.2
3.7 Ϯ 0.2
3.9 Ϯ 0.2
280,000
23,000
300,000
370,000
300,000
Atypical variant (D70G)
J variant (E497V)
J/K variant (E497V/A539T)
K variant (A539T)
10 Ϯ 2
8 Ϯ 1
13 Ϯ 3

Cocaine Hydrolase Activity of Butyrylcholinesterase
89
since His-438 is part of the catalytic triad. By contrast, Tyr- preceding formation of the acyl-enzyme intermediate is con-
328 has pushed the methyl ester away from His-438 in Fig.
5B, thus reducing the steric hindrance on His-438 and ex-
plaining the higher catalytic rate of the A328Y mutant. Our
finding that the A328F mutant also has a higher kcat for
(Ϫ)-cocaine is consistent with this model.
sistent with the idea of distinct binding and catalytic regions.
People with Genetic Variants of BChE Are At Risk.
One out of 2500 Americans and Europeans is homozygous for
atypical BChE (Whittaker, 1986; Lockridge, 1990; Kalow and
Grant, 1995). People with atypical BChE (D70G) are unable
to breathe for 2 h after a normal dose of the muscle relaxant
succinylcholine, a dose that is intended to paralyze for 3 to 5
min (Kalow and Gunn, 1957). Our results suggest that people
with atypical BChE are also at risk for cocaine intoxication.
The prediction is that a dose of cocaine tolerated by most
people will be toxic to individuals with atypical BChE. This
prediction is based on the observation that atypical BChE
(D70G) has a 10-fold lower binding affinity for (Ϫ)-cocaine
and a 10-fold lower kcat/Km value. This means that atypical
BChE is 10-fold less efficient at hydrolyzing (Ϫ)-cocaine.
Similar predictions have been made by others based on the
observation of reduced cocaine hydrolysis by plasma with low
BChE activity (Jatlow et al., 1979) or by plasma with atypical
BChE (Kalow and Grant, 1995; Schwartz and Johnson,
1996). The present work is the first to measure Km and kcat
values for atypical BChE and (Ϫ)-cocaine. A comparison with
data in the literature is listed in Table 6.
Rapid Rate of Hydrolysis of (؉)-Cocaine. The struc-
tural difference between (ϩ)- and (Ϫ)-cocaine is the location
of the methyl ester group on the tropane ring. (ϩ)-Cocaine
was hydrolyzed by BChE almost 2000-fold faster than (Ϫ)-
cocaine, the kcat values being 7500 minϪ1 and 3.9 minϪ1
,
respectively. Figure 5C shows that (ϩ)-cocaine fit easily into
the active site of wild-type BChE. The absence of steric ob-
struction of His-438 probably explains why (ϩ)-cocaine is
rapidly hydrolyzed by BChE. The A328Y mutant hydrolyzed
(ϩ)-cocaine at the same rate as wild-type BChE, a result
consistent with the model in Fig. 5C, where residue 328 is far
from the methyl ester.
Distinct Binding and Catalytic Regions. Binding af-
finities for the two enantiomers of cocaine are nearly identi-
cal (Gatley, 1991; Berkman et al., 1997; present work),
whereas rates of hydrolysis differ 2000-fold. This suggests
that the binding region may not be the same as the catalytic
region. A similar conclusion was reached by Berkman et al.
(1997) in studies with phosphonothiolates corresponding to
the transition state analogs of (Ϫ)- and (ϩ)-cocaine hydroly-
sis, when they found that the position of the methyl ester
group on the tropane ring was the determinant for hydrolysis
but not the determinant for binding efficiency. The model by
Kalow and Grant (1995) point out that people with atypical
BChE are not the only ones at risk for cocaine intoxication.
Any person with reduced BChE activity is at risk. “Any
decrease of BChE activity that prolongs the normal half-life
of cocaine would automatically favor accumulation of cocaine
Masson et al. (1997) of three enzyme-substrate complexes in blood and tissues, particularly if there is repeated cocaine
Fig. 5. Cocaine docked in the active
site of BChE. A, (Ϫ)-cocaine in wild-
type BChE. B, (Ϫ)-cocaine in the
A328Y mutant of BChE. C, (ϩ)-co-
caine in wild-type BChE.
TABLE 6
Binding and hydrolysis constants for (Ϫ)-cocaine and human BChEa
BChE
Km
Ki
kcat
Reference
pH
Temp.
␮M
␮M
minϪ1
C
Wild-type
44
7
Stewart et al. (1979)
Gatley (1991)
Mattes et al. (1996)
Schwartz and Johnson (1996)
Berkman et al. (1997)
Present work
7.4
7.2
7.4
7.0
7.4
7.0
7.0
7.0
37
30
37
Wild-type
9
Wild-type
12
6
Wild-type
37
8
14
212
744
Wild-type
25
30
30
Wild-type
14
172
4
4
Atypical (D70G)
Atypical (D70G)
Present work
Schwartz and Johnson (1996)
a The kcat of 7 was calculated from the Vmax per milliliter of serum in Stewart et al. (1979) by assuming the BChE concentration in serum was 60 pmol/ml. A kcat of 6 was
calculated from the data of Mattes et al. (1996) who reported a kcat of 24 for the tetrameric enzyme.

90
Xie et al.
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Acknowledgments
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Mary, Omaha, NE) for purifying animal BChE during her summer
rotation, Dr. B. P. Doctor (Walter Reed Army Institute of Research,
Washington DC) for procainamide-Sepharose and for the sequence of
horse BChE, Dr. G. Ghenbot (Centeon, Kankakee, IL) for purified
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Cocaine Hydrolase Activity of Butyrylcholinesterase
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