Tài liệu Báo cáo khoa học: Kinetic analysis of effector modulation of butyrylcholinesterase-catalysed hydrolysis of acetanilides and homologous esters pdf

Kinetic analysis of effector modulation of
butyrylcholinesterase-catalysed hydrolysis of acetanilides
and homologous esters
Patrick Masson
1
, Marie-The
´
re
`
se Froment
1
, Emilie Gillon
1
, Florian Nachon
1
, Oksana Lockridge
2
and
Lawrence M. Schopfer
2
1 Unite
´
d’Enzymologie, De
´
partement de Toxicologie, Centre de Recherches du Service de Sante
´
des Arme
´
es, La Tronche Cedex, France
2 University of Nebraska Medical Center, Eppley Institute, Omaha, NE, USA
Keywords

aryl acylamidase; benzalkonium;
butyrylcholinesterase; serotonin; tyramine
Correspondence
P. Masson, Unite
´
d’Enzymologie,
De
´
partement de Toxicologie, Centre de
Recherches du Service de Sante
´
des
Arme
´
es, BP 87, 38702 La Tronche Cedex,
France
Fax: +33 4 76 63 69 62
Tel: +33 4 76 63 69 59
E-mail:
(Received 30 December 2007, revised 27
February 2008, accepted 17 March 2008)
doi:10.1111/j.1742-4658.2008.06409.x
The effects of tyramine, serotonin and benzalkonium on the esterase and
aryl acylamidase activities of wild-type human butyrylcholinesterase and its
peripheral anionic site mutant, D70G, were investigated. The kinetic study
was carried out under steady-state conditions with neutral and positively
charged aryl acylamides [o-nitrophenylacetanilide, o-nitrotrifluoropheny-
lacetanilide and m-(acetamido) N,N,N-trimethylanilinium] and homologous
esters (o-nitrophenyl acetate and acetylthiocholine). Tyramine was an acti-
vator of hydrolysis for neutral substrates and an inhibitor of hydrolysis for

positively charged substrates. The affinity of D70G for tyramine was lower
than that of the wild-type enzyme. Tyramine activation of hydrolysis for
neutral substrates by D70G was linear. Tyramine was found to be a pure
competitive inhibitor of hydrolysis for positively charged substrates with
both wild-type butyrylcholinesterase and D70G. Serotonin inhibited both
esterase and aryl acylamidase activities for both positively charged and
neutral substrates. Inhibition of wild-type butyrylcholinesterase was hyper-
bolic (i.e. partial) with neutral substrates and linear with positively charged
substrates. Inhibition of D70G was linear with all substrates. A comparison
of the effects of tyramine and serotonin on D70G versus the wild-type
enzyme indicated that: (a) the peripheral anionic site is involved in the non-
linear activation and inhibition of the wild-type enzyme; and (b) in the
presence of charged substrates, the ligand does not bind to the peripheral
anionic site, so that ligand effects are linear, reflecting their sole interaction
with the active site binding locus. Benzalkonium acted as an activator at
low concentrations with neutral substrates. High concentrations of ben-
zalkonium caused parabolic inhibition of the activity with neutral sub-
strates for both wild-type butyrylcholinesterase and D70G, suggesting
multiple binding sites. Benzalkonium caused linear, noncompetitive inhibi-
tion of the positively charged aryl acetanilide m-(acetamido) N,N,N-trime-
thylanilinium for D70G, and an unusual mixed-type inhibition ⁄ activation
(a > b > 1) for wild-type butyrylcholinesterase with this substrate. No
fundamental difference was observed between the effects of ligands on
the butyrylcholinesterase-catalysed hydrolysis of esters and amides. Thus,
Abbreviations
AAA, aryl acylamidase; ASCh, acetylthiocholine; ATMA, m-(acetamido) N,N,N-trimethylanilinium; BuChE, butyrylcholinesterase; DFP,
diisopropylfluorophosphate; NATAc, N-acetylanthranilic acid; Nbs
2
, 5,5¢-dithiobis(2-nitrobenzoic acid); o-NA, o-nitroaniline; o-NAC,
o-nitroacetanilide; o-NP, o-nitrophenol; o-NPA, o-nitrophenylacetate; o-NTFNAC, o-nitrotrifluoroacetanilide; o-NTMNPA, o-N-trimethylnitro-

phenylaniline; PAS, peripheral anionic site.
FEBS Journal 275 (2008) 2617–2631 ª 2008 The Authors Journal compilation ª 2008 FEBS 2617
Cholinesterases are structurally related hydrolases [1].
Acetylcholinesterase (EC 3.1.1.7) plays a key role in
the cholinergic system in terminating the action of ace-
tylcholine, but no clear physiological function has yet
been assigned to butyrylcholinesterase (BuChE; EC
3.1.1.8) [2]. BuChE may have physiological functions
related to its esterase activity. In particular, it has been
proposed that BuChE may play a role in fatty acid [3]
and lipoprotein [4] metabolism. Studies with knock-out
mice for acetylcholinesterase indicate that BuChE can
act in the central nervous system as a surrogate acetyl-
choline-hydrolysing enzyme [5]. Both cholinesterases
also display noncholinergic activities. Cholinesterase
isoforms may have nonenzymatic roles in axonal
outgrowth, synaptogenesis, cell adhesion, neuronal
migration and developmental neurotoxicity to organo-
phosphates [6–8]. Certain nonenzymatic functions of
acetylcholinesterase have been found to depend on the
peripheral anionic site (PAS) [9]; others appear to be
related to a peptide derived from the enzyme C-termi-
nus [10]. However, the physiological relevance of these
activities is still unclear [2,11]. One noncholinergic
activity displayed by cholinesterases is aryl acylamidase
(AAA; EC 3.5.1.13) activity [12,13]. Indeed, there is
some evidence that the AAA activity of BuChE plays
a role in early brain development [14] and in the
formation of amyloid plaques in Alzheimer’s disease
[2,15].

Human plasma BuChE is of toxicological and phar-
macological importance, because it scavenges and det-
oxifies numerous carboxyl ester drugs and prodrugs
[16–18], and carbamyl and phosphoryl esters, including
nerve agents [19]. Numerous widely used chemicals are
aryl acylamides (drugs: acetaminophen, phenacetin,
flutamide, isocarboxazid, lidocaine, butanilicaine; pesti-
cide: acephate; herbicides and fungicides: acetochlor,
propanil and butachlor). The AAA activity of BuChE
in plasma and tissues could participate in the metabo-
lism of these aryl acylamide drugs and xenobiotics.
However, the potential detoxification role of the AAA
activity of BuChE needs to be addressed.
Known AAAs are serine hydrolases that catalyse the
deacylation of N-acyl arylamines [20,21]. Several
AAAs have been identified in mammalian tissues
[22,23]. Certain acryl acylamidases are identical to
carboxylesterases [24]. Albumin also displays an AAA
activity [25,26]. A correspondence between certain
molecular forms of AAAs and cholinesterases has been
demonstrated in different organs [22]. Deacetylation of
retinal melatonin into 5-methoxytryptamine is cataly-
sed by an eye AAA [27]. However, no clear physiologi-
cal function has yet been ascribed to most mammalian
AAAs. At a minimum, AAAs are toxicologically rele-
vant because they deacylate arylamide xenobiotics
[20,21,28].
The crystal structures of acetylcholinesterase and
BuChE reveal that these enzymes have a common
architecture, with only one catalytic triad located at

the bottom of a deep gorge [29]. However, it has been
suggested that esterase and amidase active centres are
nonidentical, although they are overlapping [29–33].
Contrary to this proposal, recent kinetic studies and
structure–activity relationships have clearly indicated
that BuChE utilizes the same catalytic site to hydrolyse
anilides and esters [24,25].
It has been reported that the AAA activity of
BuChE, as well as that of acetylcholinesterase, can be
either activated or inhibited by various ligands. These
ligands include: (a) biogenic amines: serotonin
(5-hydroxytryptamine), tryptamine and related mole-
cules [22,32,36–39]; (b) kynuramine [22] and tyramine
[22,36,37,39]; (c) procainamide [39]; (d) anti-Alzheimer
drugs: (+)huperzine A, donepezil, galantamine and
tacrine [32,40,41]; and (e) a cationic detergent that is
an acetylcholine (nicotinic) agonist: benzalkonium [42].
Although most ligands were found to be reversible
inhibitors of the BuChE-catalysed hydrolysis of o-nit-
roacetanilide (o-NAC), tyramine was found to be an
activator. However, kinetic analysis of these inhibiting
or activating effects was either incomplete [22,36–39]
or debatable [33,42]. In particular, reported results
were interpreted in terms of an AAA site distinct from
butyrylcholinesterase uses the same machinery, i.e. the catalytic triad
S198 ⁄ H448 ⁄ E325, for the hydrolysis of both types of substrate. The differ-
ences in response to ligand binding depend on whether the substrates are
neutral or positively charged, i.e. the differences depend on the function of
the peripheral site in wild-type butyrylcholinesterase, or the absence of its
function in the D70G mutant. The complex inhibition ⁄ activation effects of

effectors, depending on the integrity of the peripheral anionic site, reflect
the allosteric ‘cross-talk’ between the peripheral anionic site and the cata-
lytic centre.
Modulation of butyrylcholinesterase catalytic activitiy P. Masson et al.
2618 FEBS Journal 275 (2008) 2617–2631 ª 2008 The Authors Journal compilation ª 2008 FEBS
the ester site. Moreover, certain of these studies were
performed using partially purified enzymes from sera
[22,36,42], biological fluids [37,43] or commercial prep-
arations [32,42] that very probably contained serum
albumin as a contaminant. Human serum albumin has
been found to display intrinsic AAA activity [25,26].
Thus, in order to provide a complete analysis and to
clarify debated issues, we investigated the effects of
tyramine, serotonin and benzalkonium on the BuChE-
catalysed hydrolysis of neutral and charged aryl acetyl
amides [o-NAC, o-nitrotrifluoroacetanilide (o-NTF-
NAC) and m-(acetamido) N,N,N-trimethylanilinium
(ATMA)] and acetyl esters [o-nitrophenylacetate
(o-NPA) and acetylthiocholine (ASCh)] under steady-
state conditions. All of these substrates give the same
acyl enzyme intermediate. Effects on wild-type human
BuChE and its PAS mutant D70G were compared.
Because the presence of contaminating proteins dis-
playing AAA activity, e.g. albumin, in the BuChE
preparation could have biased the results, experiments
were carried out on highly purified recombinant
enzymes free of albumin and any other AAAs. It was
found that there was no fundamental difference in the
mechanisms of inhibition and activation for either the
AAA or esterase activities by these ligands. In

addition, differences between the behaviour of the
wild-type enzyme and D70G were found to reflect
alterations in the binding of positively charged
substrates ⁄ ligands on PAS, regardless of the type of
substrate (acetyl amide or acetyl ester).
Results and Discussion
Action of reversible effectors on AAA activity of
BuChE
The investigation of the effects of the ligands
(tyramine, serotonin and benzalkonium) on the AAA
and esterase activities of BuChE was performed in par-
allel on wild-type enzyme and the D70G mutant. The
substrates were neutral and positively charged acetani-
lides (o-NAC and ATMA) and esters (o-NPA and
ASCh).
The hydrolysis of neutral substrates by BuChE, in
the absence of effectors, obeys the Michaelis–Menten
model (Scheme 1, boxed mechanism in Scheme 2) that
is described by Eqn (1):
v ¼
k
cat
½E½S
K
m
þ½S
ð1Þ
with
K
m

¼
K
s
k
3
ðk
2
þ k
3
Þ
¼
K
s
½1 þðk
2
=k
3
Þ
ð2Þ
k
cat
¼
k
2
k
3
ðk
2
þ k
3

Þ
ð3Þ
k
cat
=K
m
¼ k
2
=K
s
ð4Þ
The hydrolysis of positively charged substrates by
BuChE, in the absence of effectors, shows either activa-
tion or inhibition by excess substrate. The BuChE-catal-
ysed hydrolysis of positively charged substrates is
conveniently described by Scheme 2 [44]. In Scheme 2,
the enzyme–substrate complex S
p
E corresponds to S
bound on PAS. Once the first substrate molecule has
bound to the catalytic binding site (ES), a second sub-
strate molecule can bind to PAS to form the ternary
complex S
p
ES. The kinetics of this scheme are described
by Eqn (5):
v ¼
k
cat
½E

1 þ K
m
=½S

1 þ b½S=K
ss
1 þ½S=K
ss

ð5Þ
where K
ss
is the dissociation constant of co mplexes S
p
E
and S
p
ES (K
ss
> K
m
). The parameter b reflects t he
efficiency with which S
p
ES forms p roducts. When b >1,
there is s ubstrate a ctivation; when b < 1, there is substrate
inhibition; when b = 1, the enzyme kinetics obey the
simple Michaelis–Menten model (Eqn 1). BuChE shows
substrate activation w ith ATMA (b = 1.53, K
ss

=0.7
mm [35]) and ASCh (b =2.7,K
ss
= 0.6 [45]).
The ligands act as either inhibitors or activators
depending on the nature of the substrate: neutral or
charged. Homologous pairs of substrates (e.g. acetyl
anilide ⁄ acetyl ester) show the same type of inhibition.
The binding constants for D70G were generally higher
than those for wild-type BuChE, indicating that PAS
is involved in some of these effects. Additional com-
plexities are seen with benzalkonium. The results are
summarized in Tables 1 and 2. The following is an
analysis of these effects.
Scheme 1. General scheme for hydrolysis of neutral substrates by
BuChE.
Scheme 2. General scheme for hydrolysis of positively charged
substrates by BuChE.
P. Masson et al. Modulation of butyrylcholinesterase catalytic activitiy
FEBS Journal 275 (2008) 2617–2631 ª 2008 The Authors Journal compilation ª 2008 FEBS 2619
Effects of tyramine
Tyramine was found to be an activator of both D70G-
and wild-type BuChE-catalysed hydrolysis for neutral
substrates (b >1,a > 0) (Table 1), but was an inhibi-
tor of hydrolysis for charged substrates (a =0)
(Table 2). The affinity of wild-type BuChE for tyra-
mine was higher than that of D70G with both acetyl
anilides and acetyl esters.
Our results with o-NPA and o-NAC confirm the
reports that tyramine is an activator for the hydrolysis

of o-NAC by wild-type BuChE [22,36,37,39]. The acti-
vating effect of tyramine yields the expected hyperbolic
Dixon and Cornish–Bowden plots (Fig. 1A,B) for
hydrolysis of both o-NPA and o-NAC. This nonessen-
tial activation can be mathematically treated in a man-
ner similar to partial mixed-type inhibition (see
Experimental procedures, Scheme 3). Similar activating
effects on the BuChE-catalysed hydrolysis of o-NPA
have been reported for the positively charged ligands
dibucaine [45], amiloride [46] and tetraalkylammonium
compounds [47]. This activation was interpreted in
Table 1. Effect of ligands (tyramine, serotonin and benzalkonium) on esterase and AAA activities of BuChE with the neutral substrates o-
NPA versus o-NAC. Values are means ± standard error from three to five independent determinations. H, hyperbolic; L, linear; P, parabolic.
These terms refer to the appearance of the Dixon plots. Hyperbolic curves appear when there is partial inhibition or when there is activation.
Parabolic curves indicate multiple ligand binding. A, activation; C, competitive; I, inhibition. ND, not determined.
Substrate
Tyramine Serotonin Benzalkonium
o-NPA o-NAC o-NPA o-NAC o-NPA o-NAC
Wild-type: I or A type HA HA HCI LCI PCI HA + PCI
K
a
(mM) 1 ± 0.3 0.8 ± 0.2 – – –
b
0.03 ± 0.01
K
i
(mM) – – 1.7 ± 0.5 7.7 ± 0.2 0.18 ± 0.02 0.37 ± 0.03
a ND (< 1) 0.4 1.9 ± 1.2 – – –
b 2.8 5.5 0.6 ± 0.3 ) ––
b ⁄ a ND (> 3) 14 ± 6 0.3 ± 0.3 – – –

D70G: I or A type LA LA LCI LCI HA + PCI HA + PCI
d
K
a
(mM) 9.1 ± 1.9 ND
a
––ND
c
0.015 ± 0.015
K
i
(mM) – – 12.5 ± 1 7.9 ± 0.4 0.2 ± 0.08 (> 0.3)
d
a ND (< 1) ND (> 1) 0 0 – –
b >1 >1 0 0 – –
a
No binding up to 4 mM tyramine; weak activation beyond 4 mM.
b
No activation even at low [benzalkonium].
c
Weak activation at low
[benzalkonium] at low [o-NPA].
d
Competitive inhibition occurs beyond 0.3 mM benzalkonium at the lowest [o-NAC].
Table 2. Effect of ligands (tyramine, serotonin and benzalkonium) on esterase and AAA activities of BuChE with the positively charged sub-
strates ASCh versus ATMA. Values are the means ± standard error from three to five independent determinations. H, hyperbolic; L, linear.
These terms refer to the appearance of the Dixon plots. Hyperbolic curves appear when there is partial inhibition or when there is activation.
A, activation; C, competitive; I, inhibition; M, mixed; N, noncompetitive; U, uncompetitive.
Substrate
Tyramine Serotonin Benzalkonium

ASCh ATMA ASCh ATMA ASCh
a
ATMA
Wild-type: I or A type LCI LCI LCI LUI LMI
b
LMI
c
K
a
(mM) –––––ND
d
K
i
(mM) 0.78 ± 0.07 2.4 ± 0.9 0.53 ± 0.19 0.09 ± 0.03 1.03 lM
b
0.05 lM
d
a 0 0 0 0 4.52
b
5.7
d
b 00000
b
3.1
D70G I or A type LCI LCI LCI LNI ND
a
LNI
K
a
(mM) ––––––

K
i
(mM) 9.65 ± 0.7 6.15 ± 1.8 2.74 ± 0.35 0.27 ± 0.03 ND
a
13 ± 2 lM
a 0000–0
b 0000–0
a
Benzalkonium chloride precipitated with Nbs
2
under our assay conditions.
b
At high substrate concentration [42].
c
Under experimental con-
ditions, but theory predicts HA at high substrate concentration because a > b >1.
d
Inhibition at substrate concentration lower than [S]
cross
([S]
cross
= 0.66 mM) and activation at [S] > [S]
cross
.
Modulation of butyrylcholinesterase catalytic activitiy P. Masson et al.
2620 FEBS Journal 275 (2008) 2617–2631 ª 2008 The Authors Journal compilation ª 2008 FEBS
terms of binding of the positively charged ligands to
PAS. This would form a ternary complex, L
PAS
ES,

that linearly accelerates catalysis (bk
cat
with b > 1).
The degree of activation in the presence of tyramine
was higher for the hydrolysis of o-NAC than for the
hydrolysis of o-NPA. This was determined from the
nonactivated and asymptotic limits in the nonlinear
hyperbolic acceleration plots (Fig. 1A,B), which pro-
vided estimates of b. For o-NPA hydrolysis b = 2.8,
and for o-NAC hydrolysis b = 5.5. For o-NAC,
because the hydrolysis kinetics were performed under
first-order conditions, the b ⁄ a ratio was determined
using Eqn (14) (see Experimental procedures): b ⁄ a =
14 ± 6 and a = 0.4 (Table 1). For o-NPA, experi-
ments were performed at [S] close to K
m
, so that
Eqn (13) (see Experimental procedures), which
describes velocity, gives inaccurate values for a
(a < 1) and therefore b ⁄ a >3.
The difference in the extent of activation can be
explained by differences in the rate-limiting steps.
Because the rate-limiting step for the hydrolysis of
o-NAC is acylation [35], it follows that the activating
effects of tyramine take place at the level of acylation.
For the hydrolysis of o-NPA, both acylation and deac-
ylation are partly rate limiting [35]. If, by analogy with
its effect on o-NAC, the activating effects of tyramine
reflect the acceleration of acylation, the activation of
hydrolysis of o-NPA should become limited by the

deacylation rate. This predicts that activation should
result in a modest increase in activity. Because the dif-
ference between the rates for acylation and deacylation
must be greater for o-NAC, the activation would be
expected to be greater, matching the observations
obtained (Table 1).
The binding of tyramine to D70G, in the presence
of o-NPA, is weaker (9.1-fold) than binding to the
wild-type enzyme, and induces an activating effect on
o-NPA hydrolysis (Table 1). However, it was not pos-
sible to determine the a and b parameters by nonlinear
fitting of Eqn (13). The activating effect on o-NAC
hydrolysis is apparent only for tyramine concentrations
greater than 4 mm. It is so small that it cannot be
quantified. It is clear, however, that there is a reduc-
tion in affinity with D70G for this ligand. This sup-
ports the hypothesis that PAS plays a role in binding
of this ligand to BuChE.
It was found that tyramine inhibited the turnover of
the positively charged substrates ASCh and ATMA in
a linear competitive manner (Table 2). The affinity of
tyramine for wild-type BuChE, in this inhibitory
capacity (0.78–2.4 mm), is essentially the same as its
affinity for the wild-type enzyme in its activating
capacity for neutral substrates (0.8–1.0 mm). The same
is true for the binding of tyramine to D70G, although
the affinity of D70G for tyramine is weaker than the
affinity of the wild-type enzyme. From the data in
Tables 1 and 2, it can reasonably be stated that, for
each enzyme form, K

i
= K
a
This strongly suggests that
[Tyramine] m
M
1n(1/ΔA
λ 410nm
),min
0
10
20
30
40
50
60
A

B
[Tyramine] m
M
0 1 2 3 4 5 6 0 1 2 3 4 5 6
[S]/n
0
2
4
6
8
10
12

14
16
S = o-NPA
S = o-NPA
[T
y
ramine] m
M
0 1 2 3 4
1/n(1/ΔA
λ 430nm
),min
0
100
200
300
400
500
600
S = o-NAC
Fig. 1. Activating effect of tyramine on
wild-type BuChE-catalysed hydrolysis of
o-NPA and o-NAC in 0.1
M phosphate buffer
at 25 °C. (A) o-NPA (d, 0.1 m
M; s, 0.2 mM;
, 0.4 mM; h, 0.6 mM): left panel, Dixon
plots of v
)1
versus [tyramine]; right panel,

Cornish–Bowden plots of [S] ⁄ v versus [tyra-
mine]. Nonlinear Dixon plots are expected
for activation. (B) o-NAC (d,1m
M; s,
2m
M; , 3.5 mM; h,5mM); Dixon plots of
v
)1
versus [tyramine].
P. Masson et al. Modulation of butyrylcholinesterase catalytic activitiy
FEBS Journal 275 (2008) 2617–2631 ª 2008 The Authors Journal compilation ª 2008 FEBS 2621
both the competitive inhibition of hydrolysis of posi-
tively charged substrates and activation of hydrolysis
of neutral substrates result from tyramine binding to
PAS. Such qualitatively opposite effects can be tenta-
tively interpreted in terms of allosteric inhibition ⁄ acti-
vation: the binding of tyramine to PAS induces a
conformational change that affects the formation of
the productive enzyme–substrate complex. It should be
remembered that PAS and the binding locus (W82) of
the active site are connected through an W loop
[29,44]. For positively charged substrates, the confor-
mational change prevents the productive binding of
substrate, probably by disrupting the W82–p-cation
interaction; in contrast, for neutral substrates, the con-
formational change optimizes the enzyme–substrate
orientation in the active site pocket for acylation.
Effects of serotonin
It was found that serotonin inhibited both esterase and
AAA activities of BuChE (Tables 1 and 2), in contrast

with previous reports [22]. The inhibition of wild-type
BuChE was partially (hyperbolic) competitive with the
neutral substrate o-NPA (Fig. 2A). It was linearly
competitive with o-NAC (Fig. 2B) and with the posi-
tively charged substrates ASCh and ATMA (data not
shown). The inhibition of D70G was linear with all
four substrates. The affinity of D70G for serotonin
was generally lower than that of the wild-type enzyme:
7.3-fold in the presence of o-NPA, 5.2-fold with ASCh,
three-fold with ATMA and unaffected with o-NAC.
The actual binding site of serotonin cannot be inferred
from these results. Although serotonin can bind to the
active site binding locus (W82), binding to PAS cannot
be ruled out. If serotonin binds only to W82, the affin-
ity differences between wild-type BuChE and D70G
mutant for this ligand could reflect differences in the
conformational plasticity of the active site gorge of
these enzymes.
Effects of benzalkonium on o-NPA and o-NAC
hydrolysis
The hydrolysis of both o-NPA and o-NAC by wild-
type BuChE displayed complexities in the presence of
benzalkonium. The hydrolysis of o-NAC was activated
at low benzalkonium concentration, and then inhibited
as the benzalkonium concentration increased (Fig. 3;
Table 1). There was no activation phase for the hydro-
lysis of o-NPA. Inhibition was parabolic and partial.
This biphasic behaviour suggests at least two binding
sites for benzalkonium. Activation of the BuChE-catal-
ysed hydrolysis of o-NAC by low concentrations of

benzalkonium has been reported previously [42]. With
o-NPA and o-NAC, benzalkonium shows parabolic
competitive inhibition. Parabolic inhibition suggests
that the binding of more than one benzalkonium con-
tributes to the inhibition (Fig. 4). The multiplicity of
cation binding sites was revealed with phenox-
azine ⁄ phenothiazine dyes for wild-type BuChE [48],
[Serotonin] mM
0
10
20
30
40
50
A

B
[Serotonin] mM
0 2 4 6 8 10 12 14 16
0 2 4 6 8 10 12 14 16
[S]/n
[S]/n
0
1
2
3
4
5
6
7

S = o-NPA
[Serotonin] m
M
1/n (1/ΔA
λ
410mn
), min
1/n (ΔA
λ
430nm
), min
0
500
1000
1500
2000
2500
3000
3500
4000
[Serotonin] m
M
-10 -5 0 5 10 15 0 2 4 6 8 10 12
0
2000
4000
6000
8000
10000
S = o-NAC

Fig. 2. Inhibitory effect of serotonin on wild-
type BuChE-catalysed hydrolysis of o-NPA
and o-NAC in 0.1
M phosphate buffer at
25 °C. (A) o-NPA (d, 0.1 m
M; s, 0.2 mM; ,
0.4 m
M; h, 0.6 mM; , 0.8 mM): left panel,
Dixon plots of v
)1
versus [serotonin]; right
panel, Cornish–Bowden plots of [S] ⁄ v
versus [serotonin]. Nonlinear plots indicate
partial inhibition. (B) o-NAC (d,2m
M; s,
4m
M; ,10mM): left panel, Dixon plots of
v
)1
versus [serotonin]; right panel, Cornish–
Bowden plots of [S] ⁄ v versus [serotonin].
Converging Dixon plots and parallel
Cornish–Bowden plots indicate competitive
inhibition.
Modulation of butyrylcholinesterase catalytic activitiy P. Masson et al.
2622 FEBS Journal 275 (2008) 2617–2631 ª 2008 The Authors Journal compilation ª 2008 FEBS
and with propidium for a mutant (A277W ⁄ G283D)
having PAS similar to that of acetylcholinesterase [49].
The fact that benzalkonium acts as an apparent
activator at low concentrations with o-NAC and not

with o-NPA suggests that the activation of wild-type
BuChE occurs at the level of the acylation step, similar
to the mechanism suggested for tyramine. Activation is
observable with o-NAC because acylation is rate limit-
ing (k
cat
= k
2
), whereas it is ‘buffered’ with o-NPA
because acylation and deacylation are both partly rate
limiting [35].
With D70G, benzalkonium showed a clear hyper-
bolic activation of o-NAC hydrolysis at low concentra-
tions, and parabolic inhibition at high concentrations
(Fig. 3; Table 1). There was slight activation of o-NPA
hydrolysis at low [S] and low benzalkonium concentra-
tion (data not shown). Under these conditions, the
hydrolysis kinetics are first order (cf. Eqn 12) with
k
cat
⁄ K
m
= k
2
⁄ K
s
. At low [S], the ‘buffer’ contribution
of deacylation does not take place, and activation
reflects an effect on acylation (k
2

). The fact that D70G
is slightly activated at low benzalkonium concentra-
tions, whereas the wild-type enzyme is not, indicates
that b ⁄ a ‡ 1 for D70G, whereas b ⁄ a < 1 for the wild-
type enzyme. This subtle difference in behaviour
between the two enzyme forms reflects the higher
conformational plasticity of the active site gorge of
D70G compared with that of the wild-type enzyme for
acylation with neutral ester.
Effects of benzalkonium on ATMA hydrolysis
Hydrolysis of ATMA by wild-type BuChE in the pres-
ence of increasing concentrations of benzalkonium
gave unusual Lineweaver–Burk plots (Fig. 5A) in
which the lines intersected in the upper right quadrant
at 1 ⁄ [S]
cross
$ 2 ± 0.5 mm
)1
. This is consistent with
benzalkonium being an inhibitor at low substrate con-
centration and an activator as the substrate concentra-
tion is increased. The highest ATMA concentration
(0.5 mm) was below K
ss
= 0.70 mm [35], so that acti-
vation by excess substrate did not take place. This pat-
tern of inhibition has been reported previously for
decamethonium inhibition of the hydrolysis of 7-acet-
oxy-4-methylcoumarin by acetylcholinesterase [50].
The inhibition of wild-type BuChE hydrolysis of

ATMA by benzalkonium can be described by
Scheme 3 (see Experimental procedures). For this
scheme, the Lineweaver–Burk plot is given by Eqn (6).
When a > b, a > 1 and b > 1, the Lineweaver–Burk
lines intersect in the first quadrant at 1 ⁄ [S]
cross
.
1
v
¼
aK
m
V
max
½LþK
i
b½LþaK
i

1
½S
þ
1
V
max
½LþaK
i
b½LþaK
i


ð6Þ
with the coordinates of the intersecting point:
1=½S
cross
¼
b À 1
K
m
ða À bÞ
ð7Þ
1=V
max;cross
¼
a À 1
V
max
ða À bÞ
ð8Þ
This very rare situation in which ligand L is an
inhibitor at low [S] and an activator at high [S],
[benzalkonium] m
M
ΔA
430nm
/min
0.0
0.1
0.2 0.3
0.4
0.5

0.000
0.002
0.004
0.006
0.008
0.010
S = o-NAC
Fig. 3. Concentration-dependent activation and inhibition of BuChE-
catalysed hydrolysis of o-NAC (bottom curves, 1 m
M; top curves,
5m
M) by benzalkonium. Full lines, wild-type enzyme; broken lines,
D70G mutant.
[benzalkonium] mM
1/n (1/ΔA
λ430nm
), min
0
200
400
600
800
1000
1200
0 0.1 0.2
0.3 0.4
0.5
S = o-NAC
Fig. 4. Dixon plot of the inhibitory portion of the effect of benzalko-
nium on the wild-type BuChE-catalysed hydrolysis of o-NAC. The

plot shows only benzalkonium concentrations greater than 0.1 m
M.
The substrate concentration was 1 m
M o-NAC. Nonlinearity
indicates multiple binding.
P. Masson et al. Modulation of butyrylcholinesterase catalytic activitiy
FEBS Journal 275 (2008) 2617–2631 ª 2008 The Authors Journal compilation ª 2008 FEBS 2623
beyond [S]
cross
, is symmetrical to system C5 of partial
and mixed inhibition as described by Segel [51].
The values for a and b can be determined from the
re-plots of 1 ⁄ Dslope versus 1 ⁄ [L] and 1 ⁄ Dintercept ver-
sus 1 ⁄ [L] [51]; Dslope of the Lineweaver–Burk plot is
the difference between the slope at ligand concentra-
tion [L] and the slope without ligand (Eqn 9):
Dslope ¼
aK
m
ð½LþK
i
Þ
V
max
ðb½LþaK
i
Þ
À
K
m

V
max
ð9Þ
and
1
Dslope
¼
aK
i
V
max
K
m
ða À bÞ
1
½L
þ
1
K
m
bV
max
ða À bÞ
ð10Þ
when [L] ޴, the intercept on the Dslope
)1
axis in
the re-plot is bV
max
⁄ K

m
(a – b), the intercept on the
[L]
)1
axis is – b ⁄ aK
i
and the slope is aK
i
V
max
⁄ K
m
(a ) b).
Dintercept is the difference between the intercept of
the Lineweaver–Burk plot at ligand concentration [L]
and the intercept without ligand (Eqn 11):
Dintercept ¼
ð½LþaK
i
Þ
V
max
ðb½LþaK
i
Þ
À
1
V
max
ð11Þ

and
1
Dintercept
¼
aK
i
V
max
ðb À 1Þ
1
½L
þ
bV
max
ðb À 1Þ
ð12Þ
Parameter b = 3.1 was determined from the inter-
cept on the Dintercept
)1
axis re-plot, i.e. bV
max
⁄ (b ) 1).
Then, parameter a was determined from the intercept
on the Dslope
)1
axis in the re-plot of Dslope
)1
versus
[L]
)1

, and K
i
was determined from the intercept on the
[L]
)1
axis of the Dslope
)1
re-plot (Fig. 5A, inset). This
gave a = 5.7 and K
i
= 0.05 lm .
The high value of a reflects the decreased affinity of
benzalkonium for the enzyme–substrate complex. This
result is consistent with the proposal that the binding
site for benzalkonium is either at PAS or in the active
site gorge close to PAS. The binding of benzalkonium
would then have to induce a conformational change at
the active site that is responsible for the increase in k
cat
at high [S] beyond [S]
cross
. Thus, the activating effect
of benzalkonium produces an effect similar to the acti-
vation by excess substrate that has been found to be
dependent on the integrity of PAS [44,49,52,53].
Because the rate-limiting step for the hydrolysis of
ATMA is acylation (k
2
> k
3

) [35], it is probable that
activation reflects an increase in the acylation rate.
This is similar to the activating effect of tyramine
binding on the hydrolysis of o-NAC and o-NPA. The
observation that benzalkonium is an inhibitor of
BuChE-catalysed hydrolysis of ATMA at low [S] and
an activator at high [S] suggests that the conforma-
tional change induced by the occupancy of PAS is dif-
ferent at low and high [S]. This difference could be the
result of the binding of a second substrate molecule in
the gorge at high concentration, which causes a differ-
ent (activating) conformational change in the active
site. Because the rate-limiting step of BuChE-catalysed
hydrolysis of ATMA is acylation, the second substrate
molecule must bind in the active site gorge of the
enzyme already complexed with the first substrate mol-
ecule. In acetylcholinesterase, binding of an additional
1/[ATMA] mM
1/v (1/ΔA
λ290 nm
)
, min
0
1000
2000
3000
4000
5000
6000
7000

A
B
1/[ATMA] mM
1/v (1/ΔA
λ290 nm
), min
0
1000
2000
3000
4000
5000
0 2 4 6 8 10
0.0
0.2 0.4
0.6
0.8 1.0
1/[benzalkonium] µM
–10
0
10 20
0.001
0.00 3
0.004
0.005
0.00 2
1/
Δ
sl op e
wi ld t yp e

D7 0G
Fig. 5. Lineweaver–Burk plots for the inhibition of BuChE-cataly-
sed hydrolysis of ATMA (0.1–0.5 m
M) by different concentrations
of benzalkonium in 0.1
M phosphate buffer at 25 °C. (A) Wild-
type BuChE:d, no benzalkonium;s, 0.05 l
M; , 0.1 lM; h,
0.2 l
M; ¤, 0.3 lM. Inset: re-plot of Dslope
)1
of the Lineweaver–
Burk plot as a function of the reciprocal of the benzalkonium
concentration. (B) Mutant D70G:d, no benzalkonium; s,2l
M; ,
3 l
M; h,4lM.
Modulation of butyrylcholinesterase catalytic activitiy P. Masson et al.
2624 FEBS Journal 275 (2008) 2617–2631 ª 2008 The Authors Journal compilation ª 2008 FEBS
substrate molecule to PAS and in the gorge has been
shown to inhibit enzyme activity by preventing the exit
of reaction products [54,55]. In contrast, the active site
gorge of BuChE is about 200 A
˚
3
larger than that of
the acetylcholinesterase gorge, and therefore may easily
accommodate several ligands ⁄ substrates [29,56] with-
out inhibition of substrate and product traffic.
The inhibition of D70G by benzalkonium appears

to be purely noncompetitive, i.e. the lines in the
Lineweaver–Burk plot cross on the y-axis (Fig. 5B) with
K
i
=13±2lm. Thus, the affinity of D70G for ben-
zalkonium is at least 260-fold weaker than that of the
wild-type enzyme. This suggests that PAS is the binding
site for benzalkonium, and supports the proposal that
the complexity encountered with the wild-type enzyme
reflects the binding of benzalkonium to PAS.
Effects of benzalkonium on ASCh hydrolysis
Under our experimental conditions, a study of the
inhibition of ASCh hydrolysis was not possible
because 5,5¢ -dithiobis(2-nitrobenzoic acid) (Nbs
2
) pre-
cipitated with benzalkonium. However, the inhibition
of BuChE-catalysed hydrolysis of ASCh by benzalko-
nium has been reported [42]. It is unclear how these
authors avoided the precipitation problem. In that
study, the inhibition of human BuChE was found to
be of the partial mixed type. Unfortunately, the experi-
ments were performed at high substrate concentration,
in the concentration range corresponding to substrate
activation (cf. Experimental procedures, Scheme 2,
Eqn 10). Thus, the reported K
i
value (1.03 lm) [42]
probably reflects the inhibition of substrate activation.
That is, benzalkonium is probably competing with the

formation of both S
p
E and the productive ternary
complex S
p
ES (cf. Scheme 2). Under these conditions,
the formation of S
p
E and S
p
ES is governed by a high
K
ss
(K
ss
= 0.6 mm), which is about 10 times higher
than K
m
. The K
i
value of 1.03 lm reported by these
authors is 20 times higher than our K
i
value for the
inhibition of the BuChE-catalysed hydrolysis of
ATMA (at low concentrations of ATMA). This differ-
ence supports our interpretation that these authors
were observing effects related to the substrate activa-
tion portion of the mechanism and not to the primary
hydrolytic steps.

Interaction of propanil with BuChE
Propanil (3¢,4¢-dichloroacetanilide) was not hydrolysed
by wild-type BuChE under our experimental condi-
tions, i.e. [E] > [S]. Yet, propanil binds to BuChE and
linearly inhibits the hydrolysis of o-NTFNAC and
ASCh over a large substrate concentration range. Inhi-
bition constants were determined from Dixon plots
and Cornish–Bowden plots (data not shown). Propanil
is a pure competitive inhibitor (a = 0) of the BuChE-
catalysed hydrolysis of both substrates: K
i
= 0.49 ±
0.05 mm with ASCh, and K
i
= 0.74 ± 0.58 mm with
o-NTFNAC. Thus, propanil interferes with the forma-
tion of ES, but not with S
p
EorS
p
ES.
In the BuChE–ASCh complex, the choline head
group strongly interacts with W82 [29]. The fact that
propanil is a competitive inhibitor suggests that it also
binds to the p-cation binding site W82. These results
imply that other acetyl anilide substrates (i.e. o-NAC,
o-NTFNAC, ATMA) may bind to W82 in the active
centre. This would place the substrate in the BuChE–
acetanilide substrate complexes into a favourable posi-
tion for the use of the catalytic triad Ser198 ⁄

H438 ⁄ E325 to make products. The resistance of propa-
nil to hydrolysis by BuChE probably results from
electronic effects contributed by the polar chlorine
atoms in the aromatic ring that could hamper the rota-
tional flexibility of the amide bond [57]. This could
impair appropriate orientation of the carbonyl oxygen
in the oxyanion hole.
Reaction of N-acetylanthranilic acid (NATAc)
with BuChE
Hydrolysis of the negatively charged acetanilide
NATAc by BuChE was attempted. We chose this sub-
strate because it is homologous to aspirin (N-acetyl-
salicylic acid), a negatively charged acetyl ester that is
a BuChE substrate [58]. We found that BuChE, even
at high concentration ([E] = 0.03 lm), does not hydro-
lyse NATAc ([S]
max
= 0.5 mm). Moreover, NATAc
up to 1 mm did not inhibit the BuChE-catalysed
hydrolysis of ASCh (0.035–1 mm)oro-NTFNAC
(2–3 mm). Therefore, it does not appear to bind. Com-
petition of NATAc with the three selected ligands was
not investigated.
Active structure site responsible for AAA activity
It has recently been suggested that Ser224 is the nucle-
ophile involved in the hydrolysis of aryl acylamides by
BuChE [33]. However, several lines of structural evi-
dence clearly rule out this hypothesis. First, kinetic
analysis of organophosphate inhibition of the ester
and AAA activities of BuChE indicates that there is a

single nucleophilic serine, Ser198, for both activities
[35]. Second, studies on mutant forms, e.g. silent allo-
zyme and S198C ⁄ D mutants of BuChE, support the
kinetic findings with wild-type BuChE and rule out the
P. Masson et al. Modulation of butyrylcholinesterase catalytic activitiy
FEBS Journal 275 (2008) 2617–2631 ª 2008 The Authors Journal compilation ª 2008 FEBS 2625
hypothesis that a nucleophile other than Ser198 is
responsible for the AAA activity [35]. Third, inspection
of the three-dimensional structure of human BuChE
shows that Ser224 is deeply buried inside the protein,
with Oc pointing away from the surface, about 6–7 A
˚
from the bulk solvent [29] (Fig. 6A). Therefore, no
access for substrate to Ser224 is possible.
The latter problem was acknowledged by the
authors of the Ser224 proposal. However, it was
argued that the binding of ligands such as benzalkoni-
um may induce a conformational change that activates
a Ser224 ⁄ His438 ⁄ E197 triad [33]. Our present results
show that the effect of benzalkonium on the AAA
activity of BuChE can be interpreted without postulat-
ing the unmasking of an alternative nucleophile. A
conformational change of the enzyme that would give
accessibility to Ser224 is unlikely, because it would
require a large movement of the main chain and subse-
quent disorganization of the central b-sheet. Moreover,
catalysis relies on optimal angles and distances
between the nucleophile and the base in order to allow
the formation of short, strong hydrogen bonds. The
observed spatial position of Ser224 and His438 does

not allow the formation of such a short, strong hydro-
gen bond. For example, the distance between Ne of
His438 and Oc of Ser224 is 4.8 A
˚
. In addition, Ser224
is strongly locked in a dense hydrogen bond network
that is essential for the integrity of the active site. This
dense hydrogen bond network prevents any conforma-
tional change of this residue (Fig. 6B). Ser224 notably
makes a strong hydrogen bond with Glu325
(d $ 2.5 A
˚
). Moreover, Ser224 is also hydrogen
bonded to two key water molecules that are strictly
conserved in all crystal structures of BChE in com-
plexes with charged or uncharged ligands that have
been solved to date. Ligands invariably fill the pocket
near Trp82 without triggering any alteration of this
hydrogen bond network. Finally, His438 is totally
restrained because of a stabilizing interaction with
Phe398. In contrast with the observations reported for
acetylcholinesterases [59–61], no mobility of the cata-
lytic histidine of human BChE has been observed in
crystal structures. A change in the position of this cat-
alytic histidine would be necessary for the formation
of a Ser224 ⁄ His438 ⁄ E197 triad.
Conclusions
Despite the complexity of interactions between BuChE,
tyramine, serotonin and benzalkonium, no fundamen-
tal differences were found between the effects of these

compounds on the AAA and esterase activities of
human BuChE.
The concentrations of tyramine and serotonin that
activate or inhibit the AAA activity of BuChE (and
also its esterase activity) are several orders of magni-
tude higher than the concentrations of these com-
pounds that can be encountered in vivo under
physiological conditions or even during pathological
processes. The concentration of serotonin in human
plasma of normal subjects is 9 nm [62]; it is increased
several fold as a consequence of migraine headache,
schizophrenia, hypertension or carcinoid syndrome.
The concentration of tyramine in the plasma of nor-
mal subjects is about 7 nm [63]; it is increased as a
Ser198
Ser224
A
B
Ser224
Ser198
His438
Glu325
wat
wat
2.7
2.8
2.5
2.9
2.7
Fig. 6. (A) Overall view of the three-dimensional structure of

human BuChE. The solvent-accessible surface is represented by a
mesh. Helices are represented as coils and b-sheets by arrows.
Ser224 is represented by cyan balls and Ser198 is represented by
green balls with their respective Oc in red. (B) Hydrogen bond net-
work associated with Ser224 and Ser198. Participating residues are
represented as sticks and water molecules as balls. Hydrogen bond
distances are given for the catalytic triad residues Ser198 ⁄ His438 ⁄
Glu325 (green) and Ser224 (cyan).
Modulation of butyrylcholinesterase catalytic activitiy P. Masson et al.
2626 FEBS Journal 275 (2008) 2617–2631 ª 2008 The Authors Journal compilation ª 2008 FEBS
consequence of cirrhosis, and on consumption of food
containing tyramine. Tyramine poisoning causes head-
ache, hypertensive crisis and adverse interactions with
antidepressive drugs (cf. ‘cheese syndrome’), but, even
in these extreme situations, tyramine concentrations
never reach the millimolar range. In addition, BuChE
does not hydrolyse melatonin (P. Masson & M. T.
Froment, unpublished results), a neutral aryl acyla-
mide, that is a serotonin metabolite [64]. The nocturnal
peak concentration of melatonin in blood is 0.3 nm
[64]. Thus, owing to the low affinity of BuChE for
serotonin and neutral aryl acylamides, it is not
surprising that this enzyme does not contribute to the
hydrolysis of the neurohormone. Therefore, the physi-
ological, pharmacological and toxicological relevance
of tyramine and serotonin effects on BuChE activity is
questionable. In this respect, it is unlikely that the
AAA activity of BuChE could be a link between
serotonergic and cholinergic systems, as has been
suggested [38].

Exogenous aryl acylamide compounds, drugs and
xenobiotics are mostly metabolized in the liver and
other organs by amidase-carboxylesterases [20,21].
There is no carboxylesterase in human plasma
[60,65,66]. The only AAA activities present in human
plasma are those of albumin and BuChE. Both activi-
ties are very slow with acetanilides (o-NAC, o-NTF-
NAC) [26,35]. In addition, BuChE does not hydrolyse
m-nitroacetanilide [35], the fungicide propanil (m,p-di-
chlorocacetanilide) (present study) or drugs such as
acetaminophen (p-hydroxyacetanilide) and phenacetin
(p-ethoxyacetanilide) (P. Masson & M. T. Froment,
unpublished results). Although BuChE may interact
with high concentrations of these compounds, e.g. the
inhibition of AAA activity by propanil occurs at a K
i
value of about 0.6 mm, such concentrations are far
higher than would be expected to occur in blood dur-
ing treatments or intoxications, even in the most severe
cases. Therefore, it is unlikely that BuChE plays a
significant role in the metabolism of endogenous or
exogenous aryl acylamides.
Experimental procedures
Chemicals
o-NAC was obtained from Merck (Limonest, France) and
benzalkonium chloride was obtained from Interchim
(Montluc¸ on, France). Tyramine, serotonin (5-hydroxytryp-
tamine), o-NPA, o-nitrophenol (o-NP) and o-nitroaniline
(o-NA) were purchased from Sigma Chemical France (Saint
Quentin Fallavier, France). NATAc and diisopropylfluoro-

phosphate (DFP) were obtained from Acros Organics
France (Noisy-le-Grand, France). ATMA was a gift from
T. L. Rosenberry (Mayo Clinic, Jacksonville, FL, USA).
o-NTFNAC was a gift from S. Darvesh (Dalhousie Univer-
sity, Halifax, Canada). Propanil (3¢,4¢-dichloroacetanilide)
was obtained from CIL-Cluzeau (Sainte-Foy-La-Grande,
France). Other chemicals were of biochemical grade.
Enzymes
Wild-type recombinant human BuChE and the mutant
enzyme D70G were expressed in CHO-K1 cells (ATCC,
N°CCL 61, Rockville, MD, USA). Enzymes were highly
purified by anion exchange and affinity chromatography in
20 mm potassium phosphate pH 7.0 containing 1 mm
EDTA, as described previously [44,67]. The absence of con-
taminating AAAs in purified enzymes, i.e. cell carboxyles-
terases and albumin, was controlled by nondenaturing
PAGE and activity staining [26,68].
Kinetics of substrate hydrolysis in the absence
of ligand
Kinetic assays with neutral and positively and negatively
charged acetanilides and acetyl esters were carried out at
25 °Cin0.1m phosphate buffer pH 7.0, except for o-NTF-
NAC for which the buffer was 60 mm Tris ⁄ HCl pH 8.0.
The measured rates were corrected for spontaneous hydro-
lysis of the substrates. BuChE activity on a negatively
charged substrate, NATAc, was studied under the same
conditions.
Acetanilides
Kinetic analysis of BuChE AAA activity was carried out with
a positively charged acetanilide (ATMA), a negatively

charged acetanilide (NATAc) and neutral acetanilides
(o-NAC and o-NTFNAC). The stock solution of ATMA
was in phosphate buffer, that of o-NAC was in methanol,
and those of o-NTFNAC and NATAc were in 50% acetoni-
trile. Assays with ATMA were carried out according to John-
son et al. [69]. The substrate concentration ranged from
0.025 to 5 mm. The hydrolysis of ATMA was recorded for
30 min at 290 nm (e
o)NTMNPA
= 1850 m
)1
Æcm
)1
), and rate
measurements were performed on the steady-state phase [26].
Assays with NATAc were carried out according to Kolken-
brock et al. [70]. The hydrolysis of NATAc was recorded for
30 min at 293 and 325 nm. Assays with o-NAC were carried
out according to the method of Hoagland and Graf [71]. The
final methanol concentration in the assays was 5%. The sub-
strate concentration in the assays was in the range 0.05–
4 mm. Assays with o-NTFNAC were carried out according to
Darvesh et al. [34]. The final concentration of acetonitrile in
the assays was 3.5%. The hydrolysis of o-NAC was recorded
for 45 min at 430 nm for o-NA (e
o-NA
= 4000 m
)1
Æcm
)1

).
P. Masson et al. Modulation of butyrylcholinesterase catalytic activitiy
FEBS Journal 275 (2008) 2617–2631 ª 2008 The Authors Journal compilation ª 2008 FEBS 2627
The hydrolysis of o-NTFNAC was recorded at 430 nm
(e = 3954 m
)1
Æcm
)1
) for 5 min.
Acetyl esters
Kinetic analysis of the BuChE-catalysed hydrolysis of
o-NPA was followed by monitoring the rate of appearance
of o-NP at 410 nm (e
o-NP
= 3190 m
)1
Æcm
)1
). The stock
solution of o-NPA was in methanol. The substrate concen-
tration in the assays ranged from 0.05 to 3.5 mm. The final
methanol concentration in each assay was 5.3% [45].
Hydrolysis of ASCh was followed at 420 nm using the
method of Ellman et al. [72] with 0.5 mm Nbs
2
as the chro-
mogenic reagent. The substrate concentration in the assays
ranged from 0.05 to 3.5 mm.
Kinetics under steady-state conditions
Under our experimental conditions, the BuChE-catalysed

hydrolysis of o-NAP can be investigated up to saturation.
However, because of the lower solubility of o-NAC and
o-NTFNAC, studies with these substrates were carried out
at concentrations much lower than K
m
(cf. [35]). The
steady-state catalytic parameters K
m
, k
cat
, K
ss
and b were
determined by nonlinear computed fitting of Eqns (1,5)
using the sigma plot 4 program (Jandel Science, San
Raphael, CA, USA). The active site concentration [E] of
highly purified enzyme preparations was determined
according to [73] with DFP as the titrant. The active site
concentration of wild-type BuChE was 0.16 ± 0.01 lm,
and that of the D70G mutant was 0.196 ± 0.02 lm.
Effects of ligands on steady-state kinetics
The effects of the ligands (L: tyramine, serotonin and ben-
zalkonium) on the AAA and esterase activities of wild-type
BuChE and its D70G mutant were investigated. Assays
were performed in 0.1 m phosphate buffer pH 7.0 at 25 °C.
The effect of each ligand on related pairs of substrates
(neutral substrates o-NAC and o-NPA, and positively
charged substrates ATMA and ASCh) was compared. To
avoid complications caused by substrate activation with
ATMA and ASCh, assays were performed at low and inter-

mediate substrate concentrations ([S] < K
ss
). The concen-
tration of tyramine in the assays varied from 0.1 to 15 mm,
that of serotonin from 0.05 to 15 mm, and that of
benzalkonium from 0.05 lm to 0.5 mm.
The type of inhibition or activation was determined by
visual inspection of velocity versus substrate concentration
plots (at various ligand concentrations), Lineweaver–Burk
plots, and a combination of Dixon and Cornish–Bowden
plots [74]. For benzalkonium, interactions between BuChE
(E), substrate (S) and ligand (L) showed complications, but
can be depicted by Scheme 3 at low [L].
The ligand L, acting either as an inhibitor or an activa-
tor, can bind to the free enzyme (E) or the enzyme–sub-
strate complex (ES) and modify K
m
and k
cat
. K is either an
inhibition constant (K
i
) or an activation constant (K
a
).
Assuming rapid equilibrium, the velocity equation for
Scheme 3 is described by Eqn (13):
v ¼
k
cat

½E½S
aK
m
fð½LþKÞ=ðb½LþaK Þgþ½Sfð½LþaKÞ=ðb½LþaK Þg
ð13Þ
At low substrate concentrations, [S] > K
m
:
v ¼
k
cat
f1 þðb½LÞ= ðaK Þg
K
m
f1 þð½L=KÞg
½E½Sð14Þ
If a = 0, inhibition is competitive; if a = 1, inhibition is
noncompetitive; if a > 1, inhibition is mixed. If the ternary
complex ESL is nonproductive (b = 0), there is linear
mixed inhibition. If ESL is productive with b > 1, there is
activation. Activation and inhibition of mixed type are
symmetric. If ESL makes product with 0 < b < 1 and
b ⁄ a < 1, there is nonlinear hyperbolic inhibition. In hyper-
bolic activation, 0 < a < 1 and b ⁄ a > 1. More complex
situations that can be encountered, e.g. multiple binding,
are examined in the Results and Discussion section. It
should be noted that only the b ⁄ a ratio can be determined
from Eqn (14) (cf. Table 1; activation of BuChE-catalysed
hydrolysis of o-NAC by tyramine).
Kinetic constants were determined by: (a) linearizing the

Dixon ⁄ Cornish–Bowden plots [74], followed by fitting the
line with Sigma Plot 4; (b) re-plotting the slopes from
Lineweaver–Burk plots according to Segel [51], followed by
fitting the resulting line with Sigma Plot 4; or (c) fitting the
data directly to the rate equations that describe Scheme 3.
GOSA-fit, a simulated annealing based fitting software
(BioLog, Toulouse, France; ) was
used for the fitting of nonlinear inhibition ⁄ activation
kinetics only.
Effect of propanil
Propanil was tested as a possible acetanilide substrate of
BuChE. It was assayed with wild-type enzyme in the same
manner as o-NAC. Absorbance measurements were carried
out at 430 nm for 5 min. The effect of propanil (seven
Scheme 3. General scheme for nonessential activation or partial
and mixed-type inhibition of a Michaelian enzyme.
Modulation of butyrylcholinesterase catalytic activitiy P. Masson et al.
2628 FEBS Journal 275 (2008) 2617–2631 ª 2008 The Authors Journal compilation ª 2008 FEBS
concentrations ranging from 0.05 to 0.5 mm) on the
BuChE-catalysed hydrolysis of o-NTFNAC and ASCh was
examined at four concentrations of o-NTFNAC (0.1, 0.3,
0.5 and 1 mm) and seven concentrations of ASCh (0.025,
0.05, 0.075, 0.1, 0.25, 0.5 and 1 mm).
Acknowledgements
The authors thank Professor T. Rosenberry (Mayo
Clinic, Jacksonville, FL, USA) for the gift of ATMA
and Dr S. Darvesh (Dalhousie University, Halifax,
Canada) for the gift of o-NTFNAC. The authors are
grateful to Ellen Duysen and Hasmik Grigoryan
(UNMC, Eppley Institute, Omaha, NE, USA) for their

interest in this study. This work was supported by De
´
le
´
-
gation Ge
´
ne
´
rale pour l’Armement/Programme d’Etudes
Amont (DGA/PEA) No. 01 08 07 ⁄ 03 CO 010-05 to
PM.
References
1 Massoulie
´
J, Pezzementi L, Bon S, Krejci E & Vallette
FM (1993) Molecular and cellular biology of cholines-
terases. Prog Neurobiol 41, 31–91.
2 Darvesh S, Hopkins DA & Geula C (2003) Neurobiol-
ogy of butyrylcholinesterase. Nature Neurosci 4, 131–
138.
3 Clitherow JW, Mitchard M & Harper NJ (1963) The
possible biological function of pseudocholinesterase.
Nature 199, 1000–1001.
4 Kutty KM & Payne RH (1994) Serum pseudocholines-
terase and very-low-density lipoprotein metabolism.
J Clin Lab Anal 8, 247–250.
5 Mesulam MM, Guillozet A, Shaw P, Levey A, Duysen
EG & Lockridge O (2003) Acetylcholinesterase knock-
outs establish central cholinergic pathway and can use

butyrylcholinesterase to hydrolyze acetylcholine. Neuro-
science 110, 627–639.
6 Grisaru D, Sternfeld M, Eldor A, Glick D & Soreq
H (1999) Structural roles of acetylcholinesterase vari-
ants in biology and pathology. Eur J Biochem 264,
672–686.
7 Mack A & Robitzki A (2000) The key role of butyr-
ylcholinesterase during neurogenesis and neural disor-
ders: an antisense-5¢-butyrylcholinesterase study. Prog
Neurobiol 60, 607–628.
8 Jameson RR, Seidler FJ & Slotkin TA (2007) Nonenzy-
matic functions of acetylcholinesterase splice variants in
the developmental neurotoxicity of organophosphates:
chlorpyrifos, chlorpyrifos oxon, and diazinon. Environ
Health Perspect 115, 65–70.
9 Munoz FJ, Aldunate R & Inestrosa NC (1999)
Peripheral binding site is involved in the neurotrophic
activity of acetylcholinesterase. NeuroReport 10, 3621–
3625.
10 Bon CL & Greenfield SA (2003) Bioactivity of a peptide
derived from acetylcholinesterase: electrophysiological
characterization in guinea-pig hippocampus. Eur J Neu-
rosci 17, 1991–1995.
11 Cousin X, Stra
¨
hle U & Chatonnet A (2005) Are there
non-catalytic functions of acetylcholinesterases? Lessons
from mutant animal models BioEssays 27, 189–200.
12 Checler F, Grassi J & Vincent J-P (1994) Cholinesteras-
es display genuine arylacylamidase activity but are

totally devoid of intrinsic peptidase activities. J Neuro-
chem 62, 756–763.
13 Balasubramanian AS & Banumathy CD (1993) Non-
cholinergic functions of cholinesterases. FASEB J 7,
1354–1358.
14 Boopathy R & Layer PG (2004) Aryl acylamidase activ-
ity on acetylcholinesterase is high during early chicken
brain development. Prot J 23, 325–333.
15 Guillozet AL, Smiley JF, Mash DC & Mesulam MM
(1997) Butyrylcholinesterase in the life cycle of amyloid
plaques. Ann Neurol 42, 909–918.
16 Satoh T (2005) Toxicological implications of esterases –
from molecular structures to functions. Toxicol Appl
Pharmacol 207, S11–S18.
17 Potter PM & Wadkins RM (2006) Carboxylesterases –
detoxifying enzymes and targets for drug therapy. Curr
Med Chem
13, 1045–1054.
18 Liederer BM & Borchardt RT (2006) Enzymes involved
in the bioconversion of ester-based prodrugs. J Pharm
Sci 95, 1177–1195.
19 Saxena A, Sun W, Luo C, Myers TM, Koplovitz I,
Lenz DE & Doctor BP (2006) Bioscavengers for protec-
tion from toxicity of organophosphorus compounds.
J Mol Neurosci 30, 145–147.
20 Junge W & Krisch K (1975) The carboxylesteras-
es ⁄ amidases of mammalian liver and their possible sig-
nificance. CRC Crit Rev Food Sci Nutr 3, 371–434.
21 Satoh T (1987) Role of carboxylesterases in xenobiotic
metabolism. Rev Biochem Toxicol 8, 155–181.

22 George ST & Balasubramanian AS (1981) The aryl
acylamidases and their relationship to cholinesterases
in human serum, erythrocyte and liver. Eur J Biochem
121, 177–186.
23 Hsu LL (1982) Brain aryl acylamidase. Int J Biochem
14, 1037–1042.
24 Matsushima M, Inoue H, Ichinose M, Tsukada S, Miki
K, Kurokawa K, Takahashi T & Takahashi K (1991)
The nucleotide and deduced amino acid sequences of
porcine liver proline-b-naphthylamidase. Evidence
for identity with carboxylesterase. FEBS Lett 293,
37–41.
25 Manoharan I & Boopathy R (2006) Diisopropylfluoro-
phosphate-sensitive aryl acylamidase activity of fatty
P. Masson et al. Modulation of butyrylcholinesterase catalytic activitiy
FEBS Journal 275 (2008) 2617–2631 ª 2008 The Authors Journal compilation ª 2008 FEBS 2629
acid free human serum albumin. Arch Biochem Biophys
452, 186–188.
26 Masson P, Froment M-T, Darvesh S, Schopfer LM &
Lockridge O (2007) Aryl acylamidase activity of human
serum albumin with o–nitrotrifluoroacetanilide as the
substrate. J Enz Inhib Med Chem 22 , 463–469.
27 Cahill GM & Besharse JC (1989) Retinal melatonin is
metabolized within the eye of Xenopus laevis. Proc Natl
Acad Sci USA 86, 1098–1102.
28 Cassida J & Quistad GB (2004) Organophosphate
toxicology: safety aspects of non acetylcholinesterase
secondary targets. Chem Toxicol 17, 983–998.
29 Nicolet Y, Lockridge O, Masson P, Fontecilla-Camps
J-C & Nachon F (2003) Crystal structure of human

butyrylcholinesterase and of its complexes with sub-
strate and products. J Biol Chem 278, 41141–41147.
30 Majundar R & Balasubramanian AS (1984) Chemical
modification of acetylcholinesterase from eel and basal
ganglia: effect on the acetylcholinesterase and aryl
acylamidase activities. Biochemistry 23, 4088–4093.
31 Boopathy R & Balasubramanian AS (1985) Chemical
modification of the bifunctional pseudocholinesterase.
Effect on the pseudocholinesterase and aryl acylamidase
activities. Eur J Biochem 151, 351–360.
32 Costagli C & Galli A (1998) Inhibition of cholinester-
ase-associated aryl acylamidase activity by anticholines-
terase agents: focus on drugs potentially effective in
Alzheimer’s disease. Biochem Pharmacol 55, 1733–1737.
33 Rajeh RV, Biju T, Indumathi M & Boopathy R (2007)
Does aryl acyl amidase of butyrylcholinesterase have
any physiological function? In Proceedings of the IXth
International Meeting on Cholinesterases, Suzhou, China,
May 6–10, (Tsim KWK & Jiang H-L, eds) p. 32. Hong
Kong University of Science and Technology, Hong
Kong, China.
34 Darvesh S, McDonald RS, Darvesh KV, Mataija D,
Mothana S, Cook H, Carneiro KM, Richard N, Walsh
R & Martin E (2006) On the active site for hydrolysis
of aryl amides and choline esters by human cholinester-
ases. Bioorg Med Chem 14, 4586–4599.
35 Masson P, Froment M-T, Gillon E, Nachon F, Darvesh
S & Schopfer LM (2007) Kinetic analysis of butyrylcho-
linesterase-catalyzed hydrolysis of acetanilides. Biochim
Biophys Acta 1774, 1139–1147.

36 Tsujita T & Okuda H (1983) Carboxylesterases in rat
and human sera and their relationship to serum aryl
acylamidases and cholinesterases. Eur J Biochem 133,
215–220.
37 Jayanthi LD, Balasubramanian N & Balasubramanian
AS (1992) Cholinesterases exhibiting aryl acylamidase
activity in human amniotic fluid. Clin Chim Acta 205,
157–166.
38 Weitnauer E, Robitzki A & Layer PG (1998) Aryl acy-
lamidase activity exhibited by butyrylcholinesterase is
higher in chick than in horse, but much lower than in
fetal calf serum. Neurosci Lett 254, 153–156.
39 Bhanumathy CD, Rao RV & Balasubramanian AS
(1998) Serum butyrylcholinesterase of non-human
primate shows amine sensitive aryl acyl amidase and
metallopeptidase activities and characteristics similar to
those of the human serum enzyme. Indian J Biochem
Biophys 35, 146–156.
40 Darvesh S, Walsh R & Martin E (2003) Enantiomer
affects of huperzine A on the aryl acylamidase activity
of human cholinesterases. Cell Mol Neurobiol 23, 93–
100.
41 Darvesh S, Walsh R, Kumar R, Caines A, Roberts S,
Magee D, Rockwood K & Martin E (2003) Inhibition
of human cholinesterases by drugs used to treat Alzhei-
mer disease. Alzheimer Dis Assoc Disord 17
, 117–126.
42 Jaganathan L & Boopathy R (2000) Distinct effect of
benzalkonium on the esterase and aryl acylamidase
activities of butyrylcholinesterase. Bioorg Chem 28,

242–251.
43 Rao RV, Gnanamuthu C & Balasubramanian AS
(1989) Human cerebrospinal fluid acetylcholinesterase
and butyrylcholinesterase. Evidence for identity between
the serum and cerebrospinal fluid butyrylcholinesterase.
Clin Chim Acta 183, 135–146.
44 Masson P, Legrand P, Bartels CF, Froment M-T,
Schopfer LM & Lockridge O (1997) Role of aspartate
70 and tryptophan 82 in binding of succinyldithiocho-
line to human butyrylcholinesterase. Biochemistry 36,
2266–2277.
45 Masson P, Adkins S, Gouet P & Lockridge O (1993)
Recombinant human butyrylcholinesterase G390V, the
fluoride-2 variant, expressed in Chinese hamster ovary
cells, is a low affinity variant. J Biol Chem 268, 14329–
14341.
46 Cle
´
ry C, Heiber-Langer I, Channac L, David L, Balny
C & Masson P (1995) Substrate dependence of amilo-
ride- and soman-induced conformation changes of
butyrylcholinesterase as evidenced by high-pressure
perturbation. Biochim Biophys Acta 1250, 19–28.
47 Stojan J, Golicnik G, Froment M-T, Estour F &
Masson P (2002) Concentration-dependent reversible
activation–inhibition of human butyrylcholinesterase by
tetraethylammonium ion. Eur J Biochem 269, 1154–
1161.
48 Kucukkilinc T & Ozer I (2007) Multi-site inhibition of
human plasma cholinesterase by cationic phenoxazine

and phenothiazine dyes. Arch Biochem Biophys 461,
294–298.
49 Masson P, Froment M-T, Bartels C & Lockridge O
(1996) Asp70 in the peripheral anionic site of human
butyrylcholinesterase. Eur J Biochem 235, 36–48.
50 Berman HA & Leonard K (1990) Ligand exclusion on
acetylcholinesterase. Biochemistry 29, 10640–10649.
Modulation of butyrylcholinesterase catalytic activitiy P. Masson et al.
2630 FEBS Journal 275 (2008) 2617–2631 ª 2008 The Authors Journal compilation ª 2008 FEBS
51 Segel IH (1975) Enzyme kinetics. J. Wiley & Sons, New
York, NY, pp. 161–272 (reprinted in 1993, Wiley
Classic Library Edition, ISBN 0-471-30309-7).
52 Masson P, Xie W, Froment M-T, Levitsky V, Fortier
P-L, Albaret C & Lockridge O (1999) Interaction
between the peripheral site residues of human butyr-
ylcholinesterase, D70 and Y332, in binding and
hydrolysis of substrates. Biochim Biophys Acta 1433,
281–293.
53 Masson P, Xie W, Froment M-T & Lockridge O (2001)
Effects of mutations of active site residues and amino
acids interacting with the W loop on substrate activation
of butyrylcholinesterase. Biochim Biophys Acta 1544,
166–176.
54 Stojan J, Brochier L, Alies C, Colletier JP & Fournier
D (2004) Inhibition of Drosophila melanogaster acetyl-
cholinesterase by high concentrations of substrate. Eur
J Biochem 271, 1364–1371.
55 Colletier JP, Fournier D, Greenblatt HM, Sojan J,
Sussman JL, Silman I & Weik M (2006) Structural
insights into substrate traffic and inhibition in acetyl-

cholinesterase. EMBO J 25, 2746–2756.
56 Saxena A, Redman AMG, Jiang X, Lockridge O &
Doctor BP (1997) Differences in active site gorge
dimensions of cholinesterases revealed by binding of
inhibitors to human butyrylcholinesterase. Biochemistry,
36, 14642–14651.
57 Ilieva S, Hadjieva B & Galabov B (2002) Theory sup-
plemented by experiment. Electronic effects on the rota-
tional stability of the amide group in p-substituted
acetanilides. J Org Chem 67, 6210–6215.
58 Masson P, Froment M-T, Fortier P-L, Visicchio J-E,
Bartels CF & Lockridge O (1998) Butyrylcholinesterase-
catalysed hydrolysis of aspirin, a negatively charged
ester, and aspirin-related neutral esters. Biochim Biophys
Acta 1387, 41–52.
59 Millard CB, Koellner G, Ordentlich A, Shafferman A,
Silman I & Sussman JL (1999) Reaction products of
acetylcholinesterase and VX reveal a mobile histidine in
the catalytic triad. J Am Chem Soc 121, 9883–9884.
60 Kaplan D, Barak D, Ordentlich A, Kronman C, Velan
B & Shafferman A (2004) Is aromaticity essential for
trapping the catalytic histidine 447 in human acetylcho-
linesterase? Biochemistry 43, 3129–3136.
61 Ho
¨
rnberg H, Tunemalm AK & Ekstro
¨
m F (2007) Crys-
tal structures of acetylcholinesterase in complex with
organophosphorus compounds suggest that the acyl

pocket modulates the aging reaction by precluding the
formation of the trigonal bipyramidal transition state.
Biochemistry 46, 4815–4825.
62 Kereveur A, Callebert J, Humbert M, Herve P, Simon-
neau G, Launay JM & Drouet L (2000) High plasma
serotonin concentration levels in primary pulmonary
hypertension: effect of long-term epoprostenol
(prostacyclin) therapy. Arterioscler Thromb Vasc Biol
20, 2233–2239.
63 Faraj BA, Fulenwider JT & Rypins EB (1979) Tyra-
mine kinetics and metabolism in cirrhosis. J Clin Invest
64, 413–420.
64 Boutin JA, Audinot V, Ferry G & Delagrange P (2005)
Molecular tools to study melatonin pathways and
actions. Trends Pharmacol Sci 26, 412–419.
65 Li B, Sedlacek M, Manoharam I, Boopathy R, Duysen
EG, Masson P & Lockridge O (2005) Butyrylcholinest-
erase, paraoxonase, and albumin esterase but not car-
boxylesterase, are present in human plasma. Biochem
Pharmacol 70, 1673–1684.
66 Satoh T & Hosokawa M (2006) Structure, function and
regulation of carboxylesterases. Chem–Biol Interact 162,
195–211.
67 Lockridge O, Schopfer LM, Winger G & Woods JH
(2005) Large-scale purification of butyrylcholinesterase
from human plasma suitable for injection into monkeys:
a potential new therapeutic for protection against
cocaine and nerve agent toxicity. J Med Chem Biol
Radiol Def 3, doi: 10.1901/jaba.2005.3-nihms5095.
68 Jaganathan L & Boopathy R (2000) A direct method to

visualise the arylacylamidase activity on cholinesterases
in polyacrylamide gels. BMC Biochem doi: 10.1186/
1471-2091-1-3.
69 Johnson JL, Cusack B, Davies MP, Fauq A & Rosen-
berry TL (2003) Unmasking tandem site interaction in
human acetylcholinesterase. Substrate activation with a
cationic acetanilide substrate. Biochemistry 42, 5438–
5452.
70 Kolkenbrock S, Parschat K, Beermann B, Hinz HJ &
Fetzner S (2006) N–acetylanthranilate amidase from
Arthrobacter nitroguajacolicus Ru
¨
61a, an a ⁄ b–hydro-
lase-fold protein active towards aryl-acylamides and
esters, and properties of its cysteine-deficient variant.
J Bacteriol 188, 8430–8440.
71 Hoagland RE & Graf G (1971) Nitroacetanilides as
chromogenic substrate for assaying de-acetylating
activity: the isolation and partial purification of aryl
acylamidases from erepsin and tulip. Enzymologia 41,
313–319.
72 Ellman GL, Courtney KD, Andres V & Featherstone
RM (1961) A new and rapid colorimetric determination
of acetylcholinesterase activity. Biochem Pharmacol 7,
88–95.
73 Amitai G, Moorad D, Adani R & Doctor BP (1998)
Inhibition of acetylcholinesterase and butyrylcholinest-
erase by chlorpyrifos-oxon. Biochem Pharmacol 56,
293–299.
74 Cornish-Bowden A (1974) A graphical method for

determining the inhibition constants of mixed, uncom-
petitive and non-competitive inhibitors. Biochem J 137,
143–144.
P. Masson et al. Modulation of butyrylcholinesterase catalytic activitiy
FEBS Journal 275 (2008) 2617–2631 ª 2008 The Authors Journal compilation ª 2008 FEBS 2631