Hydrolysisofacetylthiocoline,o-nitroacetanilide and
o-nitrotrifluoroacetanilide byfetalbovine serum
acetylcholinesterase
Marı
´
a F. Montenegro, Marı
´
a T. Moral-Naranjo, Encarnacio
´
n Mun
˜
oz-Delgado, Francisco J. Campoy
and Cecilio J. Vidal
Departamento de Bioquı
´
mica y Biologı
´
a Molecular-A, Universidad de Murcia, Spain
Although the best defined function of cholinesterases
(ChEs) is the cleavage of acetylcholine (ACh) in syn-
aptic and nonsynaptic locations, both acetylcholines-
terase (AChE; EC 3.1.1.7) and butyrylcholinesterase
(BuChE; EC 3.1.1.8) possess the capacity to hydrolyze
o-nitroacetanilides by means of the so-called aryl
acylamidase (AAA) activity [1–3]. Despite the lack of
information on the natural substrate and physiologi-
cal significance of the amidase activity of ChEs, its
variation during zebrafish embryogenesis [4] and brain
development [5] and its possible involvement in Alzhei-
mer’s disease [6] make the amidase activity a matter of
interest.
AChE and BuChE have 53% sequence homology,
similar folding of subunits, and similar quaternary
structures. Both ChEs show a wide range of molecular
forms, which arise from transcriptional, post-transcrip-
tional and post-translational changes [7,8]. The AChE
Keywords
aryl acylamidase; chemical denaturation;
cholinesterases; kinetic parameters;
molecular forms
Correspondence
C. J. Vidal, Departamento de Bioquı
´
mica y
Biologı
´
a Molecular-A, Edificio de Veterinaria,
Universidad de Murcia, Apdo. 4021,
E-30071 Espinardo, Murcia, Spain
Fax: +34 968 364147
Tel: +34 968 364774
E-mail: cevidal@um.es
(Received 12 November 2008, revised
27 January 2009, accepted 30 January
2009)
doi:10.1111/j.1742-4658.2009.06942.x
Besides esterase activity, acetylcholinesterase (AChE) and butyrylcholinest-
erase (BuChE) hydrolyze o-nitroacetanilides through aryl acylamidase
activity. We have reported that BuChE tetramers and monomers of human
blood plasma differ in o-nitroacetanilide (ONA) hydrolysis. The homology
in quaternary structure and folding of subunits in the prevalent BuChE
species (G
H
4
) of human plasma and AChE forms offetalbovine serum
prompted us to study the esterase and amidase activities offetal bovine
serum AChE. The k
cat
⁄ K
m
values for acetylthiocholine (ATCh), ONA and
its trifluoro derivative N-(2-nitrophenyl)-trifluoroacetamide (F-ONA)
were 398 · 10
6
m
)1
Æmin
)1
, 0.8 · 10
6
m
)1
Æmin
)1
, and 17.5 · 10
6
m
)1
Æmin
)1
,
respectively. The lack of inhibition of amidase activity at high F-ONA con-
centrations makes it unlikely that there is a role for the peripheral anionic
site (PAS) in F-ONA degradation, but the inhibition of ATCh, ONA and
F-ONA hydrolysisby the PAS ligand fasciculin-2 points to the transit of
o-nitroacetalinides near the PAS on their way to the active site. Sedimenta-
tion analysis confirmed substrate hydrolysisby tetrameric 10.9S AChE. As
compared with esterase activity, amidase activity was less sensitive to guan-
idine hydrochloride. This reagent led to the formation of 9.3S tetramers
with partially unfolded subunits. Their capacity to hydrolyze ATCh and
F-ONA revealed that, despite the conformational change, the active site
architecture and functionality of AChE were partially retained.
Abbreviations
AAA, aryl acylamidase activity; ACh, acetylcholine; AChE, acetylcholinesterase; ATCh, acetylthiocholine iodide; Brij 96, polyoxyethylene
10
-
oleyl ether; BuChE, butyrylcholinesterase; BuTCh, butyrylthiocholine; BW (BW284c51), 1,5-bis(4-allyldimethylammonium phenyl)-pentan-3-
one dibromide; ChE, cholinesterase; Fas2, fasciculin-2; F-ONA, N-(2-nitrophenyl)-trifluoroacetamide; GPI, glycosyl phosphatidylinositol; Iso-
OMPA, tetraisopropyl pyrophosphoramide; Nbs
2,
5,5¢-dithiobis(2-nitrobenzoic acid); ONA, o-nitroacetanilide; PAS, peripheral anionic site.
2074 FEBS Journal 276 (2009) 2074–2083 ª 2009 The Authors Journal compilation ª 2009 FEBS
gene produces several types of mature mRNA, depend-
ing on the choice of splice acceptor site in the 3¢-region
of the primary transcript. Thus, the AChE-T (‘tailed’),
AChE-H (‘hydrophobic’) and AChE-R (‘read-
through’) mRNAs generate the three principal types of
AChE subunit, referred to as T, H, and R, which dif-
fer in their C-termini. Also, AChE subunits can have
different N-terminal regions [9]. Upon polymerization,
AChE subunits generate various molecular forms,
which can be classified as globular (G) and asymmetric
(A) forms. The globular forms may exhibit hydrophilic
(G
H
) or amphiphilic (G
A
) properties, according to the
folding of the C-terminal domain in the monomers
(G
1
) and dimers (G
2
) composed of AChE-T subunits,
the absence or presence of the hydrophobic proline-
rich membrane anchor (PRiMA subunit) in the G
4
species composed of AChE-T subunits, and the addi-
tion of glycosyl phosphatidylinositol (GPI) to the G
1
and G
2
species consisting of AChE-H subunits [7].
Crystallographic techniques and site-directed muta-
genesis [10] have revealed a deep narrow gorge in
AChE that penetrates halfway into the protein and
contains the catalytic site at 4 A
˚
from its base. About
70% of the gorge is lined by 14 aromatic residues,
which are in the various subsites involved in ACh
accommodation. These include Trp86, Tyr133, Tyr337
and Phe338 (numbers for human AChE) of the ‘anionic
subsite’, which contributes to the stabilization of the
quaternary ammonium function of the choline moiety,
and Trp236, Phe295 and Phe297 of the ‘acyl-binding
subsite’, which fits the acetyl group into a concave
hydrophobic pocket. The structural elements involved
in the fitting of ACh in the Michaelis complex, stabil-
ization of the tetrahedral intermediate and hydrolysis
are Trp86 of the anionic binding site, the ‘oxyanion
hole’ (Gly121, Gly129, Ala204), which stabilizes the
negative charge created at the carbonyl oxygen atom of
ACh in the acetylation–deacetylation process, and the
catalytic triad (Ser203, Glu334, His447), where the Ser
hydroxyl group acts as the nucleophile against the car-
bonyl group of ACh, and the Glu-His pair is crucial
for activation of the nucleophile. At the rim of the
gorge, Tyr72, Tyr124, Trp286 and Tyr341 form part of
the ‘peripheral anionic site’ (PAS), which can bind
ACh as well as fasciculin-2 (Fas2) and the second qua-
ternary ammonium of decamethonium.
Crystallography data [11] have shown that BuChE
has a catalytic triad (Ser198, Glu325, His438) near the
bottom of a 20 A
˚
gorge, which is lined by only eight
aromatic amino acid residues, an acyl-binding pocket
(including Trp231, Leu286, Phe329, and Val288), an
anionic subsite (Trp82 and Phe329), an oxyanion hole
(Gly116, Gly117, and Ala199), and a nonaromatic
guide (Asp70 and Tyr332), which pushes butyryl-
thiocholine (BuTCh) down the active site gorge.
We have previously reported that BuChEs of human
colon, kidney andserum exhibit varying amidase ⁄
esterase activity ratios [12]. The same applies for
BuChEs of chick, horse, andfetalbovineserum [13].
In liver pathologies, there is an important increase in
amidase activity of human serum BuChE [14]. The
difference between BuChE monomers and tetra-
mers in their capacity to hydrolyze o-nitroacetanilide
(ONA) and its trifluoro derivative N-(2-nitrophenyl)-
trifluoroacetamide (F-ONA) led us to attribute the
varying amidase ⁄ esterase activity ratios to the molec-
ular polymorphism of BuChE and subtle structural
differences in the subunits [12]. AChE and BuChE
subunits generate a similar range of oligomers by poly-
merization. The variable extents to which ONA and
F-ONA are hydrolyzed by G
1
and G
4
BuChE species
from human sources [12] prompted us to investigate
the esterase and amidase activities of purified fetal
bovine serum AChE. For this purpose, catalytic effi-
ciencies of AChE acting on esterase [acetylthiocholine
(ATCh)] and amidase substrates (ONA and F-ONA)
were determined. In addition, hydrolysisof the sub-
strates by different molecular forms of AChE was
compared. Finally, the spatial requirements of AChE
subunits for expressing esterase and amidase activities
were probed by their response to the protein denatur-
ant guanidine hydrochloride.
Results and Discussion
Kinetic parameters of amidase activity associated
with AChE from fetalbovine serum
Affinity-purified AChE from fetalbovine serum
expressed both amidase and esterase activities. Both
were inhibited to a similar extent by increasing concen-
trations of 1,5-bis(4-allyldimethylammonium phenyl)-
pentan-3-one dibromide (BW284c51; BW) and Fas2
(Fig. 1). This feature, and the binding of esterase and
amidase activities to antibodies (HR2) against AChE
(Fig. 1), ruled out any contribution of BuChE and
possible contaminating esterases to the hydrolysis of
F-ONA and ONA. Hence, the results shown hereafter
correspond exclusively to esterase and amidase activi-
ties of AChE.
The almost complete suppression of both amidase
and esterase activities by the PAS inhibitor Fas-2 and
the active site inhibitor BW (Fig. 1) corroborates the
involvement of the same active site in the hydrolysis
of esterase (ATCh) and amidase (ONA and F-ONA)
substrates, a fact that has been established since
M. F. Montenegro et al. Aryl acylamidase activity of AChE
FEBS Journal 276 (2009) 2074–2083 ª 2009 The Authors Journal compilation ª 2009 FEBS 2075
Moore & Hess [15] demonstrated the equal effects of
pH on K
m
and k
cat
values for esterase and amidase
activities and their irreversible inhibition by an organo-
phosphate.
Affinity-purified fetalbovineserum AChE was able to
hydrolyze 319 ± 12 nmol ONA per mg of protein per
minute (about 30 nmolÆmin
)1
ÆmL
)1
), provided that
amidase activity was assayed at 10 mm ONA. The
activity was 20-fold that reported for purified AChE
from monkey brain [16]. In this regard, it is worth
mentioning the proposal of the high level of AChE in
early brain development as the source for the abundant
amidase activity in mammalian fetalserum [5]. In
addition, some authors have implicated amidase activity
in the formation of senile plaques through embryonic-
like AChE species identified in Alzheimer brain [6].
The catalytic parameters of AChE obtained by
tracing the esterase (ATCh) and amidase (F-ONA and
ONA) substrates are given in Table 1. The results
show that fetalbovineserum AChE displays a lower
K
m
value with ATCh than with F-ONA and ONA, a
trait also observed for BuChE [17]. Although K
m
can-
not be strictly considered to be a reliable measure of
substrate affinity, the lower K
m
of AChE with ATCh
than with o-nitroacetanilides probably reflects the
obstacles encountered by amidase substrates in reach-
ing and binding to the active site, which are due in
part to their nitro group (see later). The nearly 19-fold
lower K
m
value for fetalbovineserum AChE with
F-ONA than with ONA (Table 1) suggests that the
three fluorine atoms improve accessibility and binding
of F-ONA to the acyl-binding site.
The calculated K
m
value for fetalbovine serum
AChE with ATCh as substrate (0.23 mm) is similar to
those reported for AChE ofbovine (0.22 mm) and
human (0.28 mm) erythrocytes [18], and electric tissue
Fig. 1. Esterase and amidase activities of purified fetalbovineserum AChE are due to AChE. (A) Effect of AChE inhibitors on esterase and
amidase activities offetalbovineserum AChE. Inhibition of esterase activity, assayed with 1 m
M ATCh, andof amidase activity, measured
with 10 m
M ONA and 0.3 mM F-ONA, by BW and Fas2. (B) Binding of esterase and amidase activities to antibodies against AChE bound to
protein G–agarose.
Table 1. Kinetic parameters for esterase and amidase activities of AChE purified from fetalbovine serum. In the case offetalbovine serum
AChE, the content of catalytic sites in the reaction mixture was determined with echothiophate. In assays with ATCh, the content of cata-
lytic sites was 70 ± 10 n
M, and in assays with F-ONA and ONA it was 1540 nM. The much lower hydrolysis rate of nitroacetanilides than of
ATCh makes it necessary to raise the amount of AChE in F-ONA and ONA assays for accurate determination of the extent of their hydroly-
sis. Figures represent mean values of triplicate determinations. Constant values reported for cholinesterases from other sources are included
for comparison.
Enzyme Substrate K
m
(mM) k
cat
(min
)1
) k
cat
⁄ K
m
(M
)1
Æmin
)1
) Reference
Fetal bovineserum AChE ATCh 0.23 ± 0.01 91 700 ± 3700 398 ± 34 · 10
6
This work
Fetal bovineserum AChE F-ONA 0.81 ± 0.01 14 200 ± 100 17.5 ± 0.3 · 10
6
This work
Fetal bovineserum AChE ONA 15.22 ± 2.24 12 500 ± 2400 0.8 ± 0.3 · 10
6
This work
Human AChE ATCh 0.1 400 000 4.0 · 10
9
[20]
Human BuChE BuTCh 0.018 24 000 1.3 · 10
9
[42]
Human BuChE F-ONA > 3 > 7500 2.5 · 10
6
[26]
Human BuChE ONA > 3 > 13 4.4 · 10
3
[26]
Aryl acylamidase activity of AChE M. F. Montenegro et al.
2076 FEBS Journal 276 (2009) 2074–2083 ª 2009 The Authors Journal compilation ª 2009 FEBS
of Electrophorus (0.23–0.32 mm) [19], but higher than
that for recombinant human AChE (0.1 mm) [20]. The
differences in K
m
of AChE according to animal species
and tissues could arise from changes in active site
structure, owing to amino acid sequence, quaternary
structure (G
1
,G
2
,G
4
), and post-translational changes,
such as glycosylation and incorporation of GPI
residues. These factors could also explain the species-
specific differences in the responses of brain AChE to
organophosphorus insecticides [21]. The difference
between dimeric GPI-anchored AChEs of bovine
erythrocytes andof lymphocytes in both K
m
and
extent of lectin binding [22] illustrates the impact of
oligoglycans on the kinetic behavior of AChE. The
range of ATCh concentration, the ionic strength in the
assay and the kinetic model used for fitting experimen-
tal data may also contribute to the difference in K
m
.
Regarding substrate inhibition, fetalbovine serum
AChE was inhibited by ATCh at above 2 mm, but
amidase activity was unaffected even at 5 mm F-ONA
(more than 10 times the concentrations used in stan-
dard assays; the poor solubility of F-ONA in water
prevented us from using larger concentrations). The
lack of inhibition of amidase activity at 5 mm F-ONA
makes it unlikely that there is a role for the PAS in
increasing the association rate constant. This conten-
tion is supported by the absent or weak effects of the
PAS inhibitors propidium, gallamine and decametho-
nium on hydrolysisof ONA by electric eel AChE [23].
Nevertheless, the absence of a role for the PAS in
o-nitroacetanilide binding does not necessarily imply
an alternative route by which the catalytic pocket can
be reached. In fact, the almost complete abolition of
ATCh, ONA and F-ONA hydrolysis in the presence of
Fas2 (Fig. 1), a PAS-binding inhibitor [24], demon-
strates that o-nitroacetanilides transit near the PAS on
their way to the active site. Thus, it seems that the ste-
ric blockade created by a large polypeptide such as
Fas2 at the PAS [25] would render AChE unable to
hydrolyze both esterase and amidase substrates.
The values of k
cat
in Table 1 indicate that F-ONA
and ONA are degraded byfetalbovineserum AChE
at almost the same rate, which is much lower than the
rate for ATCh. The comparable activity of AChE on
both amidase substrates contrasts with the much faster
hydrolysis of F-ONA than ONA by BuChE [12,17,26].
The calculated k
cat
value for fetalbovineserum AChE
with ATCh as the substrate (91 700 min
)1
) is much
lower than that for AChE of humans (400 000 min
)1
)
[20] and Electrophorus (350 500 min
)1
) [27]. These k
cat
values are all greater than for the hydrolysis of
F-ONA (14 200 min
)1
) and ONA (12 500 min
)1
)
(Table 1). The much lower k
cat
for AChE with aryl
acetamides than with ATCh could arise from the
greater energy required for breaking amide than ester
bonds. Nevertheless, the greater k
cat
values for AChE
(see above) than for BuChE on F-ONA and ONA
(> 7500 min
)1
and > 13 min
)1
, respectively) [26] indi-
cate that o-nitroacetanilides are hydrolyzed more
rapidly by AChE. The moderate difference in k
cat
val-
ues for AChE and BuChE with F-ONA, as compared
with the large difference with ONA, underlines the low
catalytic rate of ONA hydrolysisby BuChE, and the
need for the three fluorine atoms in F-ONA and their
activating effect on the carbonyl group for increasing
the rate of turnover by BuChE. The faster hydrolysis
of F-ONA than of ONA by BuChE has been attri-
buted to differences in the acetylation rate [26].
As regards the orientation of o-nitroacetanilides in
the active site of ChEs, it has been suggested that they
adopt a planar and rigid structure, because of their
aromatic ring, amide bond, partial electron delocaliza-
tion, and hydrogen bonding between NH and NO
)
[17,26]. In addition, molecular modeling suggests that
the rigid structure of ONA orients its NO
2
group
towards the oxyanion hole of BuChE [26]. If this is
true, its occupation by the NO
2
group would presum-
ably prevent binding and stabilization of the alkoxide
ion of the tetrahedral transition state, and conse-
quently would impair production of the acylated tran-
sition state. Although it is not known whether ONA
and F-ONA adopt the same orientation in the BuChE
active site, the lower activation energy (higher k
cat
⁄ K
m
)
with F-ONA than with ONA may reflect the contribu-
tion of the fluorine atoms in F-ONA to the stabiliza-
tion of the alkoxide moiety and further acetylation of
BuChE. As the oxyanion hole of BuChE is composed
of Gly116, Gly117, and Ala199, and the corresponding
AChE hole is composed of Gly121, Gly122, and
Ala204 [28], the faster hydrolysisof ONA by AChE
would not arise from the amino acids in the hole, but
possibly from improved accommodation and stabiliza-
tion of the negative alkoxide intermediate in the AChE
oxyanion site.
It might be thought that the narrower active site in
AChE than in BuChE would hamper ONA hydrolysis
by AChE. Nevertheless, we feel that a smaller cavity
could force the fitting and stabilization of ONA in
the acyl-binding pocket, andby this means facilitate
the production of the alkoxide group and its entry into
the oxyanion hole. The narrower space in AChE may
allow enzyme–substrate contacts that have a positive
effect on catalysis. In contrast, a more loose interaction
of ONA with the larger acyl-binding pocket of BuChE
may prevent the substrate from assuming the required
conformation for producing the alkoxide group.
M. F. Montenegro et al. Aryl acylamidase activity of AChE
FEBS Journal 276 (2009) 2074–2083 ª 2009 The Authors Journal compilation ª 2009 FEBS 2077
When k
cat
⁄ K
m
is used as a measure of catalytic effi-
ciency, the results in Table 1 indicate that AChE
works 23 times more efficiently with ATCh than with
F-ONA, and about 500 times better than with ONA.
The greater efficiency of AChE in acting on F-ONA
than on ONA confirms the profitable use of F-ONA
for assessing the amidase activity of AChE and BuChE
[12]. AChE works approximately 22-fold more effi-
ciently with F-ONA than with ONA (compare k
cat
⁄ K
m
for the two substrates in Table 1) and BuChE 570-fold
more efficiently [26]. These data agree with previous
observations showing that the electron-withdrawing
action of the three fluorine atoms on F-ONA, which
are absent on ONA, lowers the energy barrier for
breakage of the CO–NH bond by ChEs [17,26]. The
lower activation energy (570-fold higher k
cat
⁄ K
m
) for
F-ONA hydrolysis than for hydrolysisof ONA by
BuChE arises from the big jump in the turnover of
F-ONA (> 7500 min
)1
) as compared to that of ONA
(> 13 min
)1
) [26].
To summarize, the hydrolysisof ATCh, F-ONA and
ONA by AChE confirms its capacity for degrading
esterase and amidase substrates. Nevertheless, the
higher catalytic efficiency with ATCh emphasizes
substrate preference and the main physiological role of
AChE.
Expression of esterase and amidase activities by
AChE molecular forms
We have previously reported that F-ONA and ONA
are not hydrolyzed by BuChE tetramers of human gut,
so that dimers and monomers are solely responsible
for their hydrolysis [12]. In the case of plasma, whereas
ONA was principally degraded by BuChE monomers
and to a much lower extent by tetrameric species, both
BuTCh and F-ONA were hydrolyzed by monomers
and tetramers [12]. With these premises in mind, sedi-
mentation analysis was undertaken to test the behavior
of AChE forms in fetalbovine serum.
Sedimentation profiles showed that the hydrophilic
AChE tetramers (G
H
4
) are the principal species in fetal
bovine serum (Fig. 2), in agreement with previous
results [29]. Assays of amidase activity showed that, in
contrast to the BuChE tetramers of human gut and
blood plasma [12], the tetramers offetalbovine serum
had the capacity to hydrolyze ONA and F-ONA
(Fig. 2). A shoulder at 8.6S (Fig. 2) and a small peak
at 5.5S were occasionally observed in amidase activity
profiles. They probably correspond to proteolytically
trimmed tetramers and dimers, which are hardly
detected in esterase activity profiles. If so, their obser-
vation in assays with F-ONA and ONA suggested a
higher amidase ⁄ esterase activity ratio for G
2
than for
G
4
AChE, an idea in agreement with the results of
Boopathy & Layer [5], who showed higher ONA-
hydrolyzing capacity for G
1
and G
2
than for G
4
AChE
from developing chicken brain.
The capacity of G
H
4
AChE offetalbovineserum to
hydrolyze F-ONA and ONA, the ability of G
H
4
BuChE
of human plasma to degrade F-ONA but not ONA,
and the inability of G
H
4
BuChE of human gut to
degrade F-ONA and ONA, despite the structural
homology between AChE and BuChE subunits, illus-
trate how variable the hydrolysisof amidase substrates
by ChE tetramers can be. The accessibility of the cata-
lytic site to substrate would depend on the structure of
the selected substrate, different amino acids in AChE
and BuChE polypeptides, and subtle structural varia-
tions between AChE (or BuChE) subunits according
to the homotetramer source (animal species, tissue,
and biological fluid). Post-translational changes in
ChE subunits, e.g. those arising from oligoglycans,
may affect peptide backbone fluctuations ⁄ flexibility,
and by this means hamper the ability of ONA and ⁄ or
F-ONA to enter the catalytic gorge, while the ability
Fig. 2. Sedimentation patterns showing
hydrolysis of esterase and amidase
substrates byfetalbovineserum AChE.
Sucrose density fractions were collected
and assayed with ATCh, ONA, and F-ONA.
Note the shoulder in the assays with
amidase substrates and its absence in
assays with ATCh.
Aryl acylamidase activity of AChE M. F. Montenegro et al.
2078 FEBS Journal 276 (2009) 2074–2083 ª 2009 The Authors Journal compilation ª 2009 FEBS
of ATCh to enter is maintained. In addition, active site
occlusion may arise from steric hindrance by a nearby
subunit in tetramers composed of tightly packed
subunits [30]. We are currently investigating possible
differences between human AChE monomers, dimers
and tetramers in their capacity to hydrolyze esterase
and amidase substrates.
Effect of guanidine hydrochloride on esterase
and amidase activities
In agreement with our previous data [29], a dramatic
reduction of ATCh-hydrolyzing activity was observed
when fetalbovineserum AChE was exposed to 2 m
guanidine hydrochloride (Fig. 3). As expected, the ami-
dase activity was reduced to a similar extent.
Nevertheless, the remaining activity sufficed for testing
whether esterase and amidase activities differed in
sensitivity to higher guanidine hydrochloride concen-
trations. Above 2 m guanidine hydrochloride, the rate
of ATCh hydrolysis fell rapidly and that of F-ONA
degradation fell gradually, which provided a peak in
the amidase ⁄ esterase activity ratio (Fig. 3). As both
activities reside in the same protein, we believe that the
unequal amidase ⁄ esterase activity ratio could arise
from the conformational rearrangement at high guani-
dine hydrochloride concentrations, so that the struc-
tural change could generate molecules lacking esterase
activity and retaining amidase activity or, alternatively,
molecular forms with unequally impaired esterase and
amidase activities.
Fig. 4. Sedimentation profiles showing the effect of guanidine hydrochloride on the molecular distribution of esterase and amidase activities
of fetalbovineserum AChE. Samples incubated without and with 2.3
M guanidine hydrochloride were centrifuged on 5–20% sucrose gradi-
ents containing 0.5
M guanidine hydrochloride and 0.5% Brij 96, formed on top of concentrated sucrose (40%) to prevent possible AChE
aggregates from reaching the tube bottom. Esterase activity was assayed with 1 m
M ATCh (left graph) and amidase activity with 0.3 mM
F-ONA (right). Note the different scales used for control and guanidine hydrochloride-treated fetalbovineserum AChE (left axis and right
axis, respectively). The results reveal the formation of 9.3S species at the expense of native 10.5S forms, and their expression of esterase
and amidase activity.
Fig. 3. Inactivation of esterase and amidase activities offetal bovine
serum AChE exposed to guanidine hydrochloride. Top: Change in
esterase and amidase activities with increasing concentrations of
guanidine hydrochloride. Bottom: Difference in amidase ⁄ esterase
activity ratio.
M. F. Montenegro et al. Aryl acylamidase activity of AChE
FEBS Journal 276 (2009) 2074–2083 ª 2009 The Authors Journal compilation ª 2009 FEBS 2079
In our attempts to clarify this issue, kinetic con-
stants for AChE incubated with 2.2 m guanidine
hydrochloride were determined (Table 2). The change
in K
m
and k
cat
values with ATCh and F-ONA indi-
rectly indicated that the catalytic-competent molecules
had undergone a conformational rearrangement.
Nevertheless, K
m
values revealed that the change in
substrate binding for guanidine hydrochloride-exposed
AChE (Table 2) in comparison with untreated enzyme
(Table 1) is stronger for ATCh than for F-ONA. The
k
cat
⁄ K
m
value for ATCh hydrolysis was reduced 3700-
fold by guanidine hydrochloride treatment, whereas
that for F-ONA hydrolysis was reduced only 800-fold.
This observation supported a more pronounced lower-
ing of catalytic efficiency for the former substrate.
In addition, the ability of the denaturant guanidine
hydrochloride to convert AChE tetramers into dimers
[29] made it a useful tool with which to study the rela-
tionship between AAA activity and the quaternary
organization of AChE. With this aim, samples were
incubated with 2.3 m guanidine hydrochloride and
later applied to sucrose gradients containing 0.5%
polyoxyethylene
10
-oleyl ether (Brij 96). Restoration of
native quaternary structure in AChE was prevented by
adding 0.5 m guanidine hydrochloride to the gradients.
Sedimentation profiles of ATCh-degrading and
F-ONA-degrading activities gave a prevalent 9.3S peak
with a shoulder at about 10.5S (Fig. 4). This indicated
that, although a few guanidine hydrochloride-resistant
AChE molecules remained in the native tetrameric
state (10.5S), the majority of the tetramers contained
partly unfolded subunits (9.3S) [29]. Identification of
protein aggregates displaying AChE activity at the
tube bottom (Fig. 4) supported exposure of hydropho-
bic domains in AChE, a feature exhibited by proteins
in a molten globule structure [29,31]. Expression of
esterase and amidase activities by AChE tetramers
composed of partly unfolded subunits supported the
maintenance of both activities in these conditions, in
which the native conformation of the polypeptide is
severely disturbed. It is probable that tight packing of
subunits in tetrameric AChE may restrict oscillations
of the polypeptide backbone, thus contributing
to a low level of activity under such strong denaturing
conditions.
Our results are consistent with previous observations
suggesting that catalytic efficiency and inhibitor effects
are due to global fluctuations of AChE domains
[30,32], and varying conformational states of AChE
subunits in tetrameric components [33,34]. In our view,
a flexible subunit may also be required for maintaining
catalytic ability after post-transcriptional linkage of
the structural subunits proline-rich membrane anchor
(PRiMA) and collagen-like tail subunit (ColQ) to
tetramers.
In summary, the results reported herein indicate
that: (a) the use of F-ONA for measuring the AAA
activity of ChEs is advisable; (b) as for BuChE, kinetic
parameters show that AChE can degrade the amidase
substrates F-ONA and ONA less efficiently than the
esterase substrate ATCh; (c) the esterase and amidase
activities of AChE tetramers consisting of partly
unfolded subunits demonstrate the maintenance of
catalytic ability in conditions that change protein
structure; and (d) owing to the conformational plasti-
city of AChE, the tetramers maintain catalytic com-
petence even if the subunit structure differs from the
preferred one.
Experimental procedures
Materials
Fetal bovine serum, edrophonium chloride, epoxy-activated
agarose, ATCh, 5,5¢-dithiobis(2-nitrobenzoic acid) (Nbs
2
),
tetraisopropyl pyrophosphoramide (Iso-OMPA), BW, Brij
96, guanidine hydrochloride and protein markers for sedi-
mentation analysis (beef liver catalase and beef intestine
alkaline phosphatase) were all purchased from Sigma
(St Louis, MO, USA). o-Nitroaniline and ONA were
provided by Merck (Darmstadt, Germany). F-ONA was
purchased from Princeton BioMolecular Research (USA),
and Fas2 from Latoxan (Valence, France). Echothiophate
iodide was donated by Levallois-Perret (France). Pro-
tein G-agarose was provided by Boehringer Mannheim
(Germany), and the monoclonal antibody HR2 against
human brain AChE was provided by Affinity Bioreagents
(Golden, CO, USA).
Purification of AChE and assay of esterase and
amidase activities
AChE was affinity-purified from fetalbovineserum using
an edrophonium–Sepharose matrix [29]. AChE activity was
Table 2. Kinetic parameters of AChE preincubated with 2.2 M gua-
nidine hydrochloride. Prior to guanidine hydrochloride incubation,
the concentration of active sites was lower in assays with ATCh
(approximately 1750 n
M) than in those with F-ONA (about
8250 n
M). After exposure to 2.2 M guanidine hydrochloride, the
concentrations of active sites fell to 87 n
M and 412 nM, respec-
tively.
Substrate K
m
(mM) k
cat
(min
)1
) k
cat
⁄ K
m
(M
)1
Æmin
)1
)
ATCh 1.51 ± 0.12 161 ± 12.8 107 ± 17.1 · 10
3
F-ONA 0.56 ± 0.10 12 ± 2.1 21 ± 7.4 · 10
3
Aryl acylamidase activity of AChE M. F. Montenegro et al.
2080 FEBS Journal 276 (2009) 2074–2083 ª 2009 The Authors Journal compilation ª 2009 FEBS
assayed by the Ellman method as described previously [35],
using 1 mm ATCh and 0.33 mm Nbs
2
in 100 mm sodium
phosphate (pH 7.5). Purified fetalbovineserum AChE was
able to hydrolyze 574 ± 39.5 lmol of ATCh per mg
protein per min.
Normal esterase and amidase (see below) assays – but
not those shown in Fig. 1 – were performed both with and
without 10 lm BW, and in the presence of 100 lm Iso-
OMPA. The values given correspond to esterase or amidase
activities inhibited by BW. In the presence of BW, esterase
and amidase activities of purified fetalbovineserum AChE
were negligible. Nevertheless, although the samples
contained no BuChE, assays included the BuChE inhibitor
Iso-OMPA in order to maintain our established assay
method, which has been used elsewhere for measuring
samples with AChE and BuChE activities.
One milliunit (mU) of esterase activity is the amount of
enzyme that degrades one nmol of ATCh per min at room
temperature (20–25 ° C). AChE activity in fractions col-
lected from sucrose gradients was determined by a micro-
titer assay [36,37], in which case the activity is also given in
milliunits, defined as above but taking into account the
volume of sample loaded onto the gradient [12].
Amidase activity was assayed using ONA and F-ONA
[12]. In assays with ONA, sample (25–75 l L) and a variable
volume (50–0 lL) of 50 mm potassium phosphate (pH 7.0)
(to a final volume of 75 lL) were added to microwell
plates. The reaction was started by adding 10 mm ONA
(150 lL of a 15 mm solution made in the above phosphate
buffer and 2% dimethylsulfoxide), and allowed to proceed
in an oven at 37 °C. Dimethylsulfoxide was used for its
capacity to improve ONA solubility with no effect on ester-
ase or amidase activities. The release of o-nitroaniline was
recorded at 415 ⁄ 630 nm in a microplate reader (Model 680;
Bio-Rad, Hercules, CA, USA) every 15 min for 2–3 h, and
amidase activity was calculated by reference to a calibration
curve made with 1–200 lm o-nitroaniline.
In assays with F-ONA, sample (10–20 lL), 10 lLofa
7.5 mm F-ONA solution made in acetonitrile and enough
50 mm potassium phosphate (pH 7.0) for a total volume of
250 lL were mixed. The concentration of F-ONA in the
mixture was 0.3 mm, and the production of o-nitroaniline
was recorded at 25 °C. AChE activity was unaffected by
the addition of acetonitrile. Amidase activity is given in
nmol of ONA or F-ONA hydrolyzed per min (mU).
For kinetic measurements, the ATCh concentration ran-
ged from 0.02 to 10 mm, that of ONA from 1 to 12 mm,
and that of F-ONA from 0.05 to 5 mm. Catalytic parame-
ters for ATCh hydrolysis were calculated on the assump-
tion of Michaelian behavior of AChE. Thus, K
m
and V
max
were calculated by simple weighted nonlinear regression of
the Michaelis–Menten equation using the sigma plot pro-
gram. Owing to the low solubility of ONA and F-ONA in
water, and the poor affinity of AChE for them, the hydro-
lysis kinetics were first order in substrate concentration
([S] < K
m
). Therefore, kinetic parameters were calculated
by fitting the data to the Lineweaver–Burk algorithm with
a linear regression program. The concentration of active
sites in purified fetalbovineserum AChE was determined
with echothiophate iodide as tritrant [26], and the pro-
tein content was determined with the Bradford reagent
(Bio-Rad).
Absence of contaminating esterases from
affinity-purified AChE
Because of their esterase activity, paraoxonase, carboxyles-
terase andserum albumin hydrolyze ONA and F-ONA
[38,39], and their presence would therefore lead to overesti-
mations of AChE-derived and BuChE-derived amidase
activity. Unwanted esterases in samples can be detected by
comparing the effects of AChE inhibitors on the hydrolysis
of ATCh and acetanilides. Accordingly, the possible pres-
ence of contaminating esterases in fetalbovine serum
AChE was tested by examining the effects of BW and Fas2
on esterase and amidase activities. Prior to analysis, samples
were incubated for 15 min with 1 nm to 10 m m BW (final
concentration) or with 2–500 nm Fas2. In addition, the direct
relationship between esterase and amidase activities was veri-
fied by assaying amidase activity in samples immunodepleted
of AChE. With this aim, sample aliquots offetal bovine
serum AChE were incubated with varying amounts of HR2
antibody against AChE bound to protein G–agarose (up to
5 lL of antibody per 50 lL of protein G–agarose). We have
previously reported that HR2 recognizes asymmetric, tetra-
meric, dimeric and monomeric AChE of human lymph
nodes [40] and gut [12,41]. After incubation with HR2,
agarose-bound proteins were removed, and samples devoid
of AChE were assayed for amidase activity. As an excep-
tion, these three types of assay were performed without
Iso-OMPA. The use of inhibitors and antibodies allowed us
to verify the complete absence of BuChE and unwanted
esterases from affinity-purified AChE.
Velocity sedimentation analysis
Molecular components of AChE were resolved by centri-
fugation analysis and identified by their sedimentation
coefficients. Samples and sedimentation markers, catalase
(11.4S
20,w
; Svedberg units) and alkaline phosphatase (6.1S),
were centrifuged at 165 000 g (35 000 r.p.m.) for 20 h at 4°C,
on 5-20% sucrose gradients made with 0.5% Brij 96 [35].
Fractions were collected and assayed for sedimentation mark-
ers, and AChE and amidase activities [12].
Incubation with guanidine hydrochloride
Possible differences in the susceptibility of ATCh-hydro-
lyzing and F-ONA-hydrolyzing activities of AChE to the
M. F. Montenegro et al. Aryl acylamidase activity of AChE
FEBS Journal 276 (2009) 2074–2083 ª 2009 The Authors Journal compilation ª 2009 FEBS 2081
denaturant guanidine hydrochloride were tested by sample
incubation with 0.05–3.0 m guanidine hydrochloride. The
higher resistance of amidase than of esterase activity to
mild guanidine hydrochloride treatment, along with its
capacity for dissociating AChE subunits into tetramers [29],
led us to study the quaternary structure adopted by the
guanidine hydrochloride-resistant AChE molecules. For
this, fetalbovineserum AChE was incubated with 2.3 m
guanidine hydrochloride and subjected to sedimentation
analysis. Sucrose gradients contained 0.5 m guanidine
hydrochloride, 0.5% Brij 96, and 5–20% sucrose. Deposi-
tion of AChE aggregates at the tube bottom was prevented
by placing a 0.5 mL cushion of 40% sucrose on the tube
before adding the sucrose gradient. After centrifugation,
fractions were collected and assayed for esterase and ami-
dase activities, using ATCh and F-ONA as the substrates.
Acknowledgements
This research was supported by the Fondo de Investiga-
cio
´
n Sanitaria of Spain (Grant PI04 ⁄ 1504) and the
Fundacio
´
nSe
´
neca of Murcia, Spain (Grant 00636 ⁄
PI ⁄ 04). M. F. Montenegro is a holder of a scholarship
from the Fundacio
´
nSe
´
neca.
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. Hydrolysis of acetylthiocoline, o-nitroacetanilide and
o-nitrotrifluoroacetanilide by fetal bovine serum
acetylcholinesterase
Marı
´
a. BuChE
species (G
H
4
) of human plasma and AChE forms of fetal bovine serum
prompted us to study the esterase and amidase activities of fetal bovine
serum AChE. The