Kinetic characterization of cholinesterases and a therapeutically valuable cocaine hydrolase for their catalytic activities against heroin and its metabolite 6-monoacetylmorphine

Article abstract of DOI:10.1016/j.cbi.2018.08.002

As the most popularly abused one of opioids, heroin is actually a prodrug. In the body, heroin is hydrolyzed/activated to 6-monoacetylmorphine (6-MAM) first and then to morphine to produce its toxic and physiological effects. It has been known that heroin hydrolysis to 6-MAM and morphine is accelerated by cholinesterases, including acetylcholinesterase (AChE) and/or butyrylcholinesterase (BChE). However, there has been controversy over the specific catalytic activities and functional significance of the cholinesterases, which requires for the more careful kinetic characterization under the same experimental conditions. Here we report the kinetic characterization of AChE, BChE, and a therapeutically promising cocaine hydrolase (CocH1) for heroin and 6-MAM hydrolyses under the same experimental conditions. It has been demonstrated that AChE and BChE have similar k_cat values (2100 and 1840 min^?1, respectively) against heroin, but with a large difference in K_M (2170 and 120 μM, respectively). Both AChE and BChE can catalyze 6-MAM hydrolysis to morphine, with relatively lower catalytic efficiency compared to the heroin hydrolysis. CocH1 can also catalyze hydrolysis of heroin (k_cat = 2150 min^?1 and K_M = 245 μM) and 6-MAM (k_cat = 0.223 min^?1 and K_M = 292 μM), with relatively larger K_M values and lower catalytic efficiency compared to BChE. Notably, the K_M values of CocH1 against both heroin and 6-MAM are all much larger than previously reported maximum serum heroin and 6-MAM concentrations observed in heroin users, implying that the heroin use along with cocaine will not drastically affect the catalytic activity of CocH1 against cocaine in the CocH1-based enzyme therapy for cocaine abuse.

Full text of DOI:10.1016/j.cbi.2018.08.002

Chemico-Biological Interactions 293 (2018) 107–114
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Chemico-Biological Interactions
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Kinetic characterization of cholinesterases and a therapeutically valuable
cocaine hydrolase for their catalytic activities against heroin and its
metabolite 6-monoacetylmorphine
Kyungbo Kima,b, Jianzhuang Yaoa,1, Zhenyu Jina,b, Fang Zhenga,b,∗∗, Chang-Guo Zhana,b,∗
a
Molecular Modeling and Biopharmaceutical Center, USA
Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY, 40536, USA
b
A B S T R A C T
As the most popularly abused one of opioids, heroin is actually a prodrug. In the body, heroin is hydrolyzed/activated to 6-monoacetylmorphine (6-MAM) first and
then to morphine to produce its toxic and physiological effects. It has been known that heroin hydrolysis to 6-MAM and morphine is accelerated by cholinesterases,
including acetylcholinesterase (AChE) and/or butyrylcholinesterase (BChE). However, there has been controversy over the specific catalytic activities and functional
significance of the cholinesterases, which requires for the more careful kinetic characterization under the same experimental conditions. Here we report the kinetic
characterization of AChE, BChE, and a therapeutically promising cocaine hydrolase (CocH1) for heroin and 6-MAM hydrolyses under the same experimental
−
1
conditions. It has been demonstrated that AChE and BChE have similar kcat values (2100 and 1840 min , respectively) against heroin, but with a large difference in
(2170 and 120 μM, respectively). Both AChE and BChE can catalyze 6-MAM hydrolysis to morphine, with relatively lower catalytic efficiency compared to the
K
M
−1
−1
heroin hydrolysis. CocH1 can also catalyze hydrolysis of heroin (kcat = 2150 min
relatively larger K values and lower catalytic efficiency compared to BChE. Notably, the K
and K
M
= 245 μM) and 6-MAM (kcat = 0.223 min
M
M
and K = 292 μM), with
M
values of CocH1 against both heroin and 6-MAM are all much larger
than previously reported maximum serum heroin and 6-MAM concentrations observed in heroin users, implying that the heroin use along with cocaine will not
drastically affect the catalytic activity of CocH1 against cocaine in the CocH1-based enzyme therapy for cocaine abuse.
−
1
1
. Introduction
(kcat = 4.1 min
M
and K = 4.5 μM) [12,13] and its effectiveness as an
enzyme or gene therapy for cocaine abuse treatment without significant
toxicity in animal experiments [14–17].
Heroin (3,6-diacetylmorphine) is one of the drugs most commonly
co-abused by cocaine-dependent individuals [1–6]. The concurrent use
of cocaine and heroin has received increasing clinical attentions be-
cause it not only causes more serious morbid psychopathology [7,8]
and poor addiction treatment outcomes [9,10], but also considerably
increases the risk of severe drug overdose which ends in death [11]. In
our previous studies, we designed and discovered high-activity butyr-
ylcholinesterase (BChE) mutants, also known as cocaine hydrolases
Further, CocH1 truncated after amino acid 529 was fused with
human serum albumin (HSA) to prolong the biological half-life without
changing the catalytic activity of CocH1 against cocaine [18]. The HSA-
fused CocH1 (known as Albu-CocH, Albu-CocH1, AlbuBChE or TV-1380
in the literature) has been proven safe and promising for use in animals
and humans in preclinical and clinical studies [19,20], but its actual
therapeutic value for cocaine addiction treatment is still limited by the
insufficiently high catalytic activity for cocaine hydrolysis and the in-
sufficiently long biological half-life which is ∼8 h in rats [18] or
43–77 h in humans [19]. Despite of the short biological half-life, the
Phase II clinical trial using TV-1380 for cocaine addiction treatment
was performed using a once-weekly dosing schedule. Due to the rela-
tively short biological half-life, the Phase II clinical trial did not show
(
(
CocHs), that can rapidly convert naturally occurring biologically active
−)-cocaine to physiologically inactive metabolites ecgonine methyl
ester (EME) and benzoic acid. In particular, the first one of our designed
CocHs (denoted as CocH1), i.e. the A199S/S287G/A328W/Y332G
mutant, demonstrated a ∼1000-fold improved catalytic efficiency
against (−)-cocaine compared with the wild-type BChE
∗
Corresponding author. Molecular Modeling and Biopharmaceutical Center (MMBC), Chemoinformatics and Drug Design Core of CPRI, University Research
Professor, Endowed College of Pharmacy Professor in Pharmaceutical Sciences, Department of Pharmaceutical Sciences, College of Pharmacy, University of
Kentucky, 789 South Limestone Street, Lexington, KY, 40536, USA.
∗∗
Corresponding author. Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY,
40536, USA.
E-mail addresses: fzhen2@email.uky.edu (F. Zheng), zhan@uky.edu (C.-G. Zhan).
Present address: School of Biological Science and Technology, University of Jinan, Jinan 250022, China.
1
https://doi.org/10.1016/j.cbi.2018.08.002
Received 26 April 2018; Received in revised form 24 July 2018; Accepted 3 August 2018
Available online 04 August 2018
0009-2797/ © 2018 Elsevier B.V. All rights reserved.

K. Kim et al.
Chemico-Biological Interactions 293 (2018) 107–114
statistically significant efficacy of TV-1380 with the once-weekly dosing
schedule for cocaine addiction treatment [21]. Nevertheless, it has been
concluded that “Although the continued development of TV-1380 appears
unlikely, its promising clinical profile should embolden efforts to develop new
enzyme products that are capable of delivering greater catabolic activity”
were purchased from New England Biolabs. All oligonucleotides were
purchased from Eurofins MWG Operon. Vector pCMV–MCS was ob-
tained from Agilent Technologies. Chinese hamster ovary (CHO)–S cells
and FreeStyle™ CHO Expression Medium, hypoxanthine/thymidine
(HT) supplement, L-glutamine, 4–12% Tris-glycine Mini Protein Gel,
and SimpleBlue SafeStain were purchased from Life Technologies
(Carlsbad, CA). Reduction-modified protein (rmp) Protein A Sepharose
Fast Flow was ordered from GE Healthcare Life Sciences (Pittsburgh,
PA). Centrifugal filter units were ordered from Millipore (Burlington,
MA). Heroin, 6-MAM, and morphine were provided by the National
Institute on Drug Abuse (NIDA) Drug Supply Program. All other che-
micals as well as the solvents used in high-performance liquid chro-
matography (HPLC), were of HPLC grade and purchased from Sigma-
Aldrich (St. Louis, MO).
[
21] in order to be effective with the desirable once-weekly dosing
schedule for cocaine addiction treatment.
According to our most recently reported studies in various animal
models of cocaine overdose treatment using clinically relevant timing
for rescue studies, whereas a long-lasting CocH form is required for
effective treatment of cocaine addiction using a weekly dosing sche-
dule, Albu-CocH1 (or TV-1380) itself should be more appropriate for
cocaine overdose treatment [22,23]. It has been demonstrated that the
key to cocaine toxicity treatment is to accelerate cocaine metabolism.
Once cocaine is completely converted to EME and benzoic acid, the
toxicity of cocaine will be reversed for the subjects [22].
2.2. Construction of mammalian expression plasmids
Considering the frequent use of cocaine in combination with heroin
by addicts, a question is whether or not cocaine degradation by CocH1
is significantly inhibited by heroin or its metabolites 6-mono-
acetylmorphine (6-MAM) and morphine. In fact, heroin is quickly
converted to 6-MAM and then more slowly to morphine in the circu-
lating system [24–26]. Two cholinesterases, plasma BChE and ery-
throcyte acetylcholinesterase (AChE), are generally regarded as the
principal enzymes involved in both the majority of 6-MAM formation
and significant morphine production from heroin. It has been demon-
strated that 6-MAM is the prime metabolite responsible for heroin's
acute psychoactive effects (the rush) and intoxication, but the euphoria
following the rush is more due to the stimulant effects of morphine
produced from 6-MAM hydrolysis [27–30], indicating the importance
of the rates of 6-MAM formation and degradation in the onset of heroin
effects on the central nervous system. At heroin blood concentrations
CocH1 truncated after amino acid 529 was fused with human serum
albumin (HSA) for the extension of biological half-life [38]. For protein
expression in mammalian cells, the cDNA for the CocH1 (the A199S/
F227A/S287G/A328W mutant of human BChE) containing C-terminal
HSA was generated and cloned in to pCMV-MCS in our previous studies
[12,13,39]. Two expression plasmids, pCMV-BChE-Fc(WT), and pCMV-
AChE-Fc(WT), were constructed as described previously [40]. Briefly,
the C-terminal of truncated human enzyme (BChE or AChE) was ge-
netically fused to the N-terminal of the Fc portion of wild-type human
IgG (Fc(WT)) by overlapping extension PCR with Phusion DNA poly-
merase. Then, the PCR products were digested with restriction en-
donucleases Hind III and Bgl II. The gel purified PCR products were
then ligated to the pCMV-MCS expression vector using T4 DNA ligase.
2
4, 29, 31−32
attainable in vivo ≤ 270 nM
, ∼80% of the total heroin
2.3. Protein expression and purification
hydrolysis in blood is accounted for plasma and erythrocyte cytosol
where BChE and AChE are located, respectively [33–35]. In vitro en-
zyme kinetic studies using purified native human cholinesterases fur-
ther demonstrated that BChE, rather than AChE, is mainly responsible
for degradation of heroin to 6-MAM with a higher catalytic efficiency
under first-order kinetics [36]. However, there has been controversy
over the catalytic activity and functional significance of the cholines-
terases (AChE and BChE) on the hydrolysis of 6-MAM to morphine
CHO-S cells were incubated in FreeStyle CHO Expression Medium
(Life Technologies) with 8 mM L-glutamine (Life Technologies) at 37 °C
in a humidified atmosphere with 8% CO and transfected with gene
2
expression DNA constructs encoding the protein of interest using the
TransIT-PRO Transfection Kit (Mirus Bio LLC, Madison, WI) when the
6
number of the cells reached 1.0 × 10 cells/mL. The culture medium
was harvested 6 days after transfection. The Fc-fused protein (BChE or
AChE) secreted into the culture medium was purified by protein A af-
finity chromatography. After removing cells by centrifugation, the cell-
free culture medium was mixed with rmp Protein A Sepharose Fast
Flow (GE Healthcare Life Sciences) pre-equilibrated with 20 mM
Tris⋅HCl (pH 7.4) and incubated for overnight at 6 °C with occasional
stirring. Then, the suspension was packed in a column and washed with
[
1,36,37], which makes it difficult to interpret their actual roles in 6-
MAM degradation. In addition, the reported values of the kinetic
parameters (kcat and K ) for BChE against heroin ranged from 12.9 to
40 min and from 0.11 to 3.5 mM, respectively [1,36,37], requiring
M
−1
5
for the more careful kinetic characterization under the same experi-
mental conditions.
In the present study, we kinetically compared CHO cell-expressed
human recombinant AChE, BChE, and CocH1 with the aims to examine
their catalytic efficiencies against heroin and 6-MAM and to assess the
possible interaction between cocaine and heroin or 6-MAM in their
hydrolysis reactions catalyzed by CocH1 in comparison with human
enzymes AChE and BChE. The complete catalytic parameters obtained
for AChE, BChE, and CocH1 against heroin and 6-MAM reveal how the
abused drugs (cocaine and heroin) can possibly affect each other in
terms of their hydrolysis reactions and detoxification under various
conditions. The insights from the kinetic characterization will be va-
luable in guiding further development of novel enzyme therapies for the
drug detoxification. In particular, concurrent use of heroin and cocaine
is not expected to significantly affect the efficacy of CocH1 (or its fusion
protein form TV-1380) in cocaine detoxification.
5 column volume (CV) of 20 mM Tris⋅HCl (pH 7.4) until an
OD280 < 0.02 was achieved; then the protein was eluted by adjustment
of the pH and salt concentration. HSA-fused CocH1 was also expressed
as described above. Using the AlbuPure matrix (Prometic Life Sciences
Inc., Laval, Canada), CocH1-HSA was purified where the cell-free cul-
ture medium was loaded onto packed bed pre-equilibrated with 50 mM
sodium acetate (pH 5.3), extensively washed with 8 CV of equilibration
buffer. Then, the resin bound protein was eluted with 5 CV of 50 mM
ammonium acetate, pH 7.4. For buffer exchange, the eluate was dia-
lyzed in storage buffer (50 mM Hepes, 20% sorbitol, 1 M glycine, pH
7.4) by Millipore Centrifugal Filter Units. The entire purification pro-
cess was performed in a cold room at 8 °C and the purified proteins
were stored at −80 °C until the activity tests.
2.4. Enzyme activity assays
2. Materials and methods
Enzymatic hydrolyses of heroin to 6-MAM and 6-MAM to morphine
2
.1. Materials
were tested under the following assay conditions. Incubations (50 μl
final volume) contained purified enzyme and heroin or 6-mono-
acetylmorphine (6-MAM) in 0.1 M phosphate buffer, pH 7.4. All the
Phusion DNA polymerases, restriction enzymes, and T4 DNA ligase
108

K. Kim et al.
Chemico-Biological Interactions 293 (2018) 107–114
Fig. 1. Enzymatic activity of BChE and AChE on the hydrolysis of heroin and 6-MAM. Schematic presentation of heroin hydrolysis to morphine (A), and chroma-
tograms for the deacetylation of heroin (B) and 6-MAM (C) in the presence or absence of BChE or AChE. Peak 1 (morphine) with retention time 3.8 min, peak 2 (6-
MAM) with retention time 4.9 min and peak 3 (heroin) with retention time 14 min. The enzyme (AChE or BChE) was incubated with 1 mM substrate concentration at
4
μM designated enzyme at 37 °C for 25 min. Ten microliters of the incubation supernatant were injected onto HPLC for analysis.
activity assays were performed at 37 °C. For heroin hydrolysis,
mixture so that each incubation (200 μl final volume) finally contained
100 ng/ml CocH1 and 100 μM (−)-cocaine with or without 100 μM
heroin or 6-MAM in 0.1 M phosphate buffer, pH 7.4. The enzymatic
reactions occurred at 25 °C and stopped at varying time points by the
addition of 200 μl of 0.1 M HCl. Since the reaction product benzoic acid
is neutralized at strong acidic pH, whereas (−)-cocaine is protonated
on its amino nitrogen, the radioactive product was extracted by 1 ml of
toluene and measured by scintillation counting. Finally, the measured
(−)-cocaine concentration-dependent radiometric data were analyzed
to determine the residual concentrations of cocaine after the enzymatic
reactions.
0
6
.02–2.5 mM heroin was incubated with 35 nM designated enzyme. For
-MAM hydrolysis, 0.002–2 mM 6-MAM was incubated with 2 μM de-
signated enzyme. The reaction time and concentration were adjusted
such that no more than 10% of substrate was depleted during reaction.
The reaction was terminated, and protein was precipitated by the ad-
dition of 100 μl of iced 50% acetonitrile/0.5 M hydrochloric acid, fol-
lowed by 5 min centrifugation at 15,000 g. The resulting supernatants
were subjected to reverse-phase HPLC (RP-HPLC) on a 5 μm C18 110 Å
column (250 × 4.6 mm; Gemini) (Life Technologies) and RP-HPLC was
performed using the mobile phase consisting of 20% acetonitrile in
0
.1% TFA. The remaining substrate and resulting products were mon-
itored by a fluorescence detector with an excitation wavelength of
30 nm and emission wavelength of 315 nm and by monitoring UV
2.6. Molecular modelling
2
absorbance at 230 nm. The quantification was based on a standard
curve prepared using an authentic standard compound.
Heroin and 6-MAM binding with human AChE, BChE, and CocH1
were modelled by using our previously modelled structures of the same
enzymes [42–45]. Our previous molecular dynamics (MD) simulations
on the structures of enzyme-substrate complexes started from the X-Ray
crystal structures deposited in the protein databank (PDB) (AChE: code
1B41; BChE: 2XQF and 1P0P). Molecular docking and subsequent op-
timization were carried out using a similar protocol described pre-
viously [44]. Briefly, the acetyl group of the substrate (heroin or 6-
MAM) was positioned in the oxyanion hole (consisting of Gly116,
Gly117, and Ala199 in BChE, or Gly121, Gly122, and Ala204 in AChE,
or Gly116, Gly117, and Ser199 in CocH1), and the positively charged
2.5. In vitro hydrolysis of cocaine by CocH
Enzymatic hydrolysis of (−)-cocaine by CocH1 was tested under the
3
following assay conditions. A sensitive radiometric assay using [ H]
(
(
−)-cocaine labelled on its benzene ring was employed to measure
−)-cocaine and benzoic acid, the product of the enzymatic (−)-co-
3
caine hydrolysis [41]. Briefly, 100 nCi of [ H](−)-cocaine was first
mixed with heroin, 6-MAM, or water. CocH1 was then added to the
109

K. Kim et al.
Chemico-Biological Interactions 293 (2018) 107–114
amino-group of the substrate (heroin and 6-MAM) was placed in the
choline-binding site near Trp82 in BChE and CocH1 or Trp86 in AChE.
Finally, the binding models of heroin and 6-MAM in the corresponding
enzyme-substrate complexes were optimized by performing the energy
minimization.
AChE [36]. This led us to examine whether free 6-MAM molecules can
serve as a substrate for BChE or not. To address this question, the en-
zymatic activity of the enzyme (BChE or AChE) was studied using
synthetic 6-MAM which we added to the reaction system. 1 mM 6-MAM
was mixed and incubated with either 40 μM enzyme (BChE or AChE)
under the incubation condition mentioned above. The results showed
that direct incubation of 6-MAM with BChE produced the amount of
morphine which is significantly larger than that in the control (without
an enzyme), demonstrating that free 6-MAM molecules can serve as a
substrate for BChE (Fig. 1C). Interestingly, AChE also converted syn-
thetic 6-MAM to morphine in a significant amount comparable to that
of morphine produced from heroin by AChE (Fig. 1B and C). Overall,
these observations clearly indicate that both free heroin and 6-MAM
molecules produced after heroin uptake in the circulatory system can be
metabolized to morphine by both cholinesterases (AChE and BChE) in
blood.
2.7. Statistical analysis
Statistical analyses were performed with the GraphPad Prism 5.01
software (San Diego, CA). Comparisons of multiple factors were ex-
amined using the two-way ANOVA with post hoc analysis, allowing us
to examine the significance of the difference in the in vivo activity data
between each pair of the reaction systems. p < 0.05 was considered
statistically significant.
3. Results & discussion
3.1. Morphine formation from heroin in the presence of recombinant human
3.3. Kinetics of heroin hydrolysis by BChE, AChE, and CocH1
BChE and AChE
As a potential anti-cocaine medication, CocH1 has a considerably
−
1
Two previously reported studies led to contradictory findings over
the ability of BChE to catalyze hydrolysis of 6-MAM to morphine
1,36,37], which limits the interpretation of the data concerning the
improved catalytic efficiency (k = 3060 min , KM = 3.1 μM, and
cat
8 − 1 − 1
kcat/KM = 9.9 × 10 min
M
) compared to the wild-type BChE
−1
5
− 1
−
[
(k = 4.1 min , KM = 4.5 μM, and kcat/KM = 9.1 × 10 min
M
cat
1
actual contributions of the enzymes to the drug metabolism to mor-
phine in blood. In 1999, Salmon and his colleagues reported that only
AChE, but not BChE, further hydrolyzes 6-MAM to morphine from
heroin [36], but these findings are opposite to the previous observa-
tions of Kamendulis et al. showing the capability of BChE to catalyze 6-
) against (−)-cocaine. Thus, CocH1 may be used to effectively block
the drug reward for a given dose of cocaine. Considering that CocH1 is
developed from human BChE capable of metabolizing all of (−)-co-
caine, heroin and its initial host metabolite 6-MAM, (−)-cocaine de-
gradation by CocH1 can be affected by the drug-drug interaction with
heroin or 6-MAM. Specifically, if heroin or 6-MAM can also be hydro-
lyzed by CocH1, we would like to know the KM or the binding affinity
−1
MAM into morphine (kcat = 0.25 min
M
and K = 8.6 mM) [1].
Therefore, we first tested whether or not heroin is metabolized to 6-
MAM and then eventually into morphine by recombinant human BChE
or AChE. For each enzyme, 1 mM heroin was incubated with 4 μM en-
zyme. As shown in Fig. 1B, in the presence of either BChE or AChE, after
(K ) of the drug (heroin or 6-MAM) with CocH1 in order to estimate
how heroin or 6-MAM could competitively inhibit CocH1 for its cata-
lytic activity against (−)-cocaine. In principle, for a competitive in-
d
25 min of incubation, heroin has completely been converted to 6-MAM,
hibition of an enzyme, the inhibitory constant (K ) value is equal to the
i
and some 6-MAM has further been converted to morphine. Both BChE
and AChE were highly active in metabolizing heroin to 6-MAM, but
they were less active in further degrading 6-MAM to morphine. These
results clearly show that like AChE, BChE is capable of hydrolyzing
heroin to morphine eventually, which are in agreement with the find-
ings of Kamendulis et al. [1], but unlike the observations of Salmon
et al. [36]. We also observed that AChE produced more morphine than
BChE in the given reaction condition, implying the relatively lower
catalytic efficiency of BChE against 6-MAM.
corresponding Kd value (K = K ). However, the K value can be dif-
i
d
d
ferent from the corresponding KM value. Nevertheless, K ≈ K value
d
M
under the well-known rapid equilibrium assumption [46] which is
usually true for enzyme-substrate binding. Hence, we may reasonably
use an experimentally measured KM of CocH1 against heroin or 6-MAM
to estimate the potential inhibitory activity of heroin or 6-MAM against
CocH1-catalyzed hydrolysis of another substrate like (−)-cocaine when
K ≈ K .
i
M
In order to know whether heroin or 6-MAM can significantly inhibit
CocH1-catalyzed hydrolysis of (−)-cocaine, we investigated kinetics of
heroin degradation to 6-MAM by CocH1, BChE, and AChE. Under the
experimental conditions generating the kinetic data depicted in Fig. 2,
we only observed the metabolite 6-MAM, and there were no detectable
levels of morphine, indicating that the enzyme activity for converting 6-
MAM to morphine is much lower than that for converting heroin to 6-
MAM. The obtained kinetic data are depicted in Fig. 2, and the kinetic
3
.2. Hydrolysis of free 6-MAM to morphine by human recombinant BChE
and AChE
In a previous report by Salmon et al. [36], it was noted that AChE
hydrolyzes 6-MAM only when 6-MAM is produced from heroin within
its active site and free 6-MAM molecules only serve as an inhibitor for
Fig. 2. Kinetic analysis of heroin hydrolysis by BChE, AChE, and CocH1. The hydrolysis of heroin to 6-MAM by BChE (A), AChE (B), and CocH1 (C) were determined
at substrate concentrations of 0.015–1.25 mM (BChE and CocH1) or 0.015–7.5 mM (AChE). Kinetic parameters (kcat and K ) were determined by fitting the measured
M
reaction rate data to the Michaelis-Menten kinetic equation using the Prism5.01 software. Each dot is the representative of the average of triplicates and its values are
expressed as the mean ± standard deviation.
110

K. Kim et al.
Chemico-Biological Interactions 293 (2018) 107–114
Table 2
Kinetic parameters of BChE, AChE, and CocH1 against 6-MAM.
Table 1
Kinetic parameters of BChE, AChE, and CocH1 against heroin.
Enzyme
k
cat (min−1)
K
M
(μM)
kcat/K
M
(min−1 M−1
)
RCE
R2
Enzyme
k
cat (min−1
)
K
M
(μM)
kcat/K
M
(min−1 M−1
)
RCE
R2
7
3
BChE
AChE
CocH1
1840 ± 24
2100 ± 26
2150 ± 31
120 ±
2170 ±
245 ±
5
9
1.53 × 10
9.68 × 10
8.78 × 10
1
0.06
0.57
0.991
0.997
0.994
BChE
AChE
CocH1
0.065 ± 0.001
7.078 ± 0.141
0.223 ± 0.005
24 ± 1.4
259 ± 18
292 ± 22
2.71 × 10
2.73 × 10
1
0.990
0.990
0.987
5
6
4
6
10.1
0.282
3
0.764 × 10
parameters obtained are summarized in Table 1. As shown in Table 1,
compared to BChE, CocH1 has a higher K value (245 μM compared to
AChE against heroin.
M
−
1
−
1
20 μM) and a similar kcat value (2150 min
compared to 1840 min
1
3.4. Kinetics of 6-MAM hydrolysis by BChE, AChE, and CocH1
)
. The determined K of CocH1 against heroin is ∼900-fold larger
M
than the previously reported blood heroin concentrations attainable in
vivo (≤0.27 μM) [24,29,31,32] and ∼76-fold larger than its reported
As mentioned above, 6-MAM may also potentially inhibit CocH1-
catalyzed cocaine hydrolysis because 6-MAM can also serve as a sub-
strate for wild-type BChE (Fig. 1B and C). To access this possibility, we
examined the kinetics of 6-MAM degradation to morphine by CocH1 as
M
K value against (−)-cocaine (3.1 μM) [12,13]. Generally speaking, for
i
a given inhibitor, when the K value is ∼900-fold larger than the in-
hibitor concentration, the inhibitor can only decrease the enzyme ac-
tivity by less than ∼0.1%, suggesting that the blood heroin levels
usually achieved by the heroin users are not expected to significantly
change the enzymatic hydrolysis of (−)-cocaine by CocH1. Moreover,
as one can see from the kinetic data in Table 1, AChE has ∼18-fold
well as wild-type BChE and AChE. The catalytic parameters kcat and K
M
were determined for BChE against 6-MAM, and then were compared
with those of AChE and CocH1 (Table 2 and Fig. 3). As seen in Table 2,
the K
which is ∼94-fold-larger than the reported K
against (−)-cocaine. Given that the maximum serum concentration
max) of 6-MAM in humans has been reported to range from 5.2 to
7.5 μM after intravenous heroin administration [48–50], the K
(292 μM) of CocH1 against 6-MAM is still ∼16–56-fold larger than the
of 6-MAM achieved by heroin users. In comparison, the observed
M
value of CocH1 against 6-MAM was determined to be 292 μM
M
value (3.1 μM) of CocH1
larger K
cat value (2100 min
These data indicate that the major difference between wild-type AChE
M
value (2170 μM) compared to that of BChE, but with a similar
− 1 − 1
k
compared to 1840 min
), against heroin.
(
C
1
M
and BChE in the catalytic efficiency against heroin 5 (kcat
/
7
− 1 − 1
−
K
M
= 1.53 × 10 min
M
M
for BChE vs kcat/K = 9.68 × 10 min
1
− 1
C
max
M
for AChE) is mainly attributed to their difference in the binding
peak blood (−)-cocaine concentrations were ∼3-fold higher than the
of CocH1 against (−)-cocaine. Overall, these data suggest that when
affinity with heroin. Our kinetic data strongly support the argument
37,47] that plasma BChE is the prime enzyme responsible for the rapid
enzymatic hydrolysis of the 3′-phenolic ester of heroin in the blood.
In this study, our experimental K value of BChE against heroin
K
M
[
both 6-MAM and (−)-cocaine reach their corresponding peak con-
centrations in the blood, CocH1-catalyzed (−)-cocaine hydrolysis can
only be inhibited by 6-MAM for ∼0.45–1.5%. The lower the 6-MAM
concentration, the less the inhibition. The potential inhibition by 6-
MAM would not be significant. Hence, CocH1 can still efficiently de-
grade (−)-cocaine at the 6-MAM concentrations usually achieved by
heroin users. According to the kinetic data in Table 2, the determined
M
(
120 μM) is consistent with the earlier K
M
of 110 μM reported by
Lockridge et al. [1,36,37] and cited by Salmon et al. [36], but quite
different from the number of Kamendulis et al. (3.5 mM) [1,36,37]. The
−
1
catalytic rate constant (kcat = 1840 min
) determined is also sub-
−
1
stantially higher than the wide range (from 12.9 to 540 min
) re-
4
− 1 − 1
k
cat/K
M
value (2.73 × 10 min
M
) of AChE against 6-MAM was
ported by those research groups [1,36,37]. Moreover, the kinetic
parameter values of AChE determined for the hydrolysis of heroin to 6-
3
− 1 − 1
approximately 10 times greater than that (2.71 × 10 min
M
) of
−
1
BChE against 6-MAM. Notably, these findings are in agreement with the
findings of Kamendulis et al. [1,36,37] in that BChE catalyzes the hy-
MAM (kcat = 2100 min
values (kcat = 351 min
[
and K
and K = 620 μM) reported by Salmon et al.
36]. It is likely that the differences in the catalytic parameters de-
M
= 2170 μM) are higher than the
−
1
M
drolysis of 6-MAM into morphine. However, our experimental K
cat values are much different from the corresponding kinetic para-
meters reported by Kamendulis et al. [1,36,37]. Our experimental K of
4 μM is considerably smaller than their K of 8.6 mM and our de-
termined catalytic rate constant (kcat = 0.065 min ) is also much
M
and
k
termined for the same enzymatic reactions are largely dependent on
how enzymes are prepared if all of the kinetic assays are all in readily
controlled experimental conditions. Generally, natural protein sources,
especially from human or animal tissues, have the difficulty to meet the
requirements for higher retention of functional properties including
their enzymatic activities, mainly due to the complicated collection,
treatment, storage, and extraction processes. These processes may affect
the protein structure accompanied with a change (usually a decrease) in
its binding affinity and activity, and result in inactivation or overall
diminished enzymatic activity. Whereas the kinetic studies of Lockridge
et al. [1,36,37], Salmon et al. [36], and Kamendulis et al. [1,36,37]
were found on the use of natural BChE or AChE extracted from human
blood samples, all the kinetic analysis in the present study were per-
formed using the freshly expressed and purified BChE and AChE for
comparison. In addition, another external factor affecting the enzy-
matic reaction kinetics is the temperature. The kinetic studies of
Salmon et al. [36], and Kamendulis et al. [1,36,37] were performed at
M
2
M
−1
−
1
different from the earlier kcat of 0.25 min . Accounting for all of the
kinetic parameters, the catalytic efficiency
= 2.71 × 10 min M−1) obtained for the same enzymatic hy-
(kcat
/
3
−1
K
M
drolysis in the present study is ∼93-fold larger than that (kcat
/
1
−1 −1
K
M
= 2.91 × 10 min
M
) reported by Kamendulis et al. [1,36,37].
As mentioned above, the differences in the catalytic parameters de-
termined for the same enzymatic reaction seem to largely rely on how
BChE is prepared for the kinetic assay. Compared to our kinetic analysis
using the freshly expressed and purified BChE, the previous kinetic
analysis by Kamendulis et al. [1,36,37] was based on the use of natural
BChE extracted from human plasma samples.
Overall, all of our experimental kinetic data strongly suggest that
BChE and AChE play distinct roles in heroin metabolism into morphine.
BChE catalyzes hydrolysis of heroin to 6-MAM with a much higher
catalytic efficiency than AChE. For the further degradation of 6-MAM to
morphine, BChE has a relatively lower catalytic efficiency than AChE.
Further, we tested whether (−)-cocaine degradation by CocH1 will
be affected by the drug-drug interaction when heroin or 6-MAM is
present in the reaction system. According to the results obtained de-
picted in Fig. 4, (−)-cocaine degradation by CocH1 was not sig-
nificantly changed in the presence of even an abnormally high
3
7 °C, but that of Lockridge et al. [1,36,37] was accomplished at 25 °C.
In this study, the same enzymatic kinetic analysis was carried out at
7 °C and the protein samples used this study are shown to have higher
binding affinity and catalytic efficiency (reflected by the lower K and
higher kcat/K , respectively). These observations strongly suggest that
3
M
M
the kinetic parameters determined in the present study are more likely
to reasonably reflect the actual enzymatic activity of human BChE and
111

K. Kim et al.
Chemico-Biological Interactions 293 (2018) 107–114
Fig. 3. Kinetic analysis of 6-MAM hydrolysis by BChE, AChE, and CocH1. The hydrolysis of heroin to 6-MAM by BChE (A), AChE (B), and CocH1 (C) were determined
) were determined by fitting the generated reaction rate data to the Michaelis-Menten
at substrate concentrations of 5–2000 μM. Kinetic parameters (kcat and K
M
kinetic equation using the Prism5.01 software. Each dot is the representative of the average of triplicates and its values are expressed as the mean ± standard
deviation.
enzyme (AChE, BChE, or CocH1). The molecular docking enabled us to
understand how heroin may bind with human AChE, BChE, and
CocH11 compared to 6-MAM binding with the corresponding enzymes.
According to the enzyme-substrate binding structures obtained from
molecular docking (as shown in Fig. 5), the binding mode for each
enzyme (AChE, BChE, and CocH1) with heroin is similar to that with 6-
MAM in terms of the overall hydrogen bonding with the oxyanion hole,
particularly for the crucial interactions between the carbonyl oxygen of
the substrate and the oxyanion hole of the enzyme. In particular, there
are always two hydrogen bonds between the carbonyl oxygen of the
substrate and backbone amide groups of the oxyanion hole residues
(
Gly120 and Gly121 of AChE corresponding to Gly116 and Gly117 of
BChE and CocH11) no matter whether the substrate is heroin or 6-
MAM. There are also two hydrogen bonds between the carbonyl oxygen
of the substrate and the oxyanion hole of CocH1 (sidechain of Ser199
and backbone of Gly117), no matter whether the substrate is heroin or
6-MAM.
The modelling results depicted in Fig. 4 reveal that, no matter
whether the enzyme is AChE, BChE, or CocH1, the acetyl-group of
heroin forms stronger hydrogen bonds in the oxyanion hole compared
to 6-MAM with the same enzyme. Hence, all of the enzymes concerned
in the present study are expected to have a significantly better catalytic
efficiency against heroin compared to 6-MAM. In particular, although a
novel hydrogen bond from Ser199 of CocH1 is introduced to stabilize
the acetyl-group of the substrate (heroin or 6-MAM), the hydrogen
bond with Gly116 is lost compared to wild-type BChE. Therefore,
CocH1 concerned in the present study is not expected to have a sig-
nificantly improved catalytic efficiency against heroin or 6-MAM in
comparison with BChE. The computational insight is supported by the
measured kinetic parameters discussed above.
Fig. 4. (−)-Cocaine hydrolysis by CocH1. Both the enzyme (100 ng/ml) and
−)-cocaine (100 μM) were incubated together with 100 μM opioid (heroin or
-MAM). Presented are the residual concentrations of cocaine versus time (○, no
(
6
enzyme control; ■, only enzyme control, without opioid; , enzyme plus
heroin; , enzyme plus 6-MAM). Cocaine concentrations were determined by
3
using sensitive radiometric assays using [ H](−)-cocaine. Results represent two
independent experiments and the values are expressed as mean ± standard
deviations.
4. Conclusion
concentration (100 μM) of heroin or 6-MAM.
The catalytic activities of wild-type AChE, wild-type BChE, and
3.5. Insights from molecular modeling
CocH1 against heroin and 6-MAM have been characterized under the
same experimental conditions for comparison. According to the de-
The reaction pathways of cholinesterase-catalyzed hydrolyses of
termined kinetic parameters kcat and K
M
for all of these enzymatic re-
heroin and 6-MAM were studied in our previous computational studies
44,45] through first-principles quantum mechanics and molecular
actions, wild-type AChE and BChE have similar kcat values (kcat = 2100
−1
−1
[
min
for AChE and kcat = 1840 min
for BChE) against heroin.
mechanics-free energy (QM/MM-FE) simulations. The optimized re-
actant complexes (e.g. BChE complexed with heroin and 6-MAM, and
AChE complexed with 6-MAM) obtained from our previous QM/MM
studies show a couple of important enzyme-substrate interaction fea-
tures. One is the interaction between the acetyl groups of heroin and 6-
MAM and the oxyanion hole consisting of Gly116, Gly117, and Ala199
in BChE (corresponding to Gly121, Gly122, and Ala204 in AChE). The
other is the interaction between the positively charged amino-groups of
heroin and 6-MAM and the sidechain of Trp82 in BChE (corresponding
to Trp86 in AChE). Guided by the aforementioned interactions, the
substrate (heroin or 6-MAM) was docked into the active site of the
However, BChE has a ∼16 fold-higher catalytic efficiency than AChE
7
−
1
−
1
(kcat/K
= 9.67 × 10 min
∼18-fold stronger binding affinity with heroin compared to AChE
(K ≈ K ≈ K = 2170 μM for AChE).
M
= 1.53 × 10 min
M
for
BChE
vs
cat
k /
5
− 1
M− 1 for AChE), mainly because BChE has a
K
M
d
M
= 120 μM for BChE vs
K
d
M
Besides, both AChE and BChE can catalyze 6-MAM hydrolysis to mor-
phine, with relatively lower catalytic efficiency compared to the cor-
responding enzyme catalyzing heroin hydrolysis.
−
1
CocH1 can also catalyze hydrolysis of heroin (kcat = 2150 min
−
1
and
K
M
= 245 μM) and 6-MAM (kcat = 0.223 min
and
K
M
= 292 μM), with relatively larger K
M
values and relatively lower
112

K. Kim et al.
Chemico-Biological Interactions 293 (2018) 107–114
Fig. 5. Modeled structures of the AChE, BChE, and CocH1 (E14-3) binding with heroin and 6-MAM. (A) Wild-type AChE binding with heroin; (B) Wild-type BChE
binding with heroin; (C) CocH1 binding with heroin; (D) Wild-type AChE binding with 6-MAM; (E) Wild-type BChE binding with 6-MAM; (F) CocH1 binding with 6-
MAM.
catalytic efficiency compared to wild-type BChE. Notably, the K
M
va-
through grant CHE-1111761. The authors also acknowledge the
Computer Center at University of Kentucky for supercomputing time on
a Dell X-series Cluster with 384 nodes or 4768 processors.
lues of CocH1 against both heroin and 6-MAM are all much larger than
previously reported maximum serum heroin and 6-MAM concentrations
observed in heroin users, implying that the heroin use along with co-
caine use will not significantly affect the catalytic activity of CocH1
against cocaine in the CocH1-based enzyme therapy for cocaine abuse.
Appendix A. Supplementary data
Supplementary data related to this article can be found at https://
doi.org/10.1016/j.cbi.2018.08.002.
Acknowledgements
This work was supported in part by the National Institutes of Health
through the NIDA Translational Avant-Garde Award (UH2/UH3
DA041115) and R01 grants (R01 DA035552, R01 DA032910, R01
DA013930, and R01 DA025100) and the National Science Foundation
Transparency document
Transparency document related to this article can be found online at
https://doi.org/10.1016/j.cbi.2018.08.002.
113

K. Kim et al.
Chemico-Biological Interactions 293 (2018) 107–114
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