Báo cáo khoa học: Insights into substrate and product traffic in the Drosophila melanogaster acetylcholinesterase active site gorge by enlarging a back channel ppt

Insights into substrate and product traffic in the
Drosophila melanogaster acetylcholinesterase active site
gorge by enlarging a back channel
Florian Nachon
1
, Jure Stojan
2
and Didier Fournier
3
1De
´
partement de Toxicologie, CRSSA, Grenoble, France
2 Institute of Biochemistry, Medical Faculty, Ljubljana, Slovenia
3 IPBS, Universite
´
Paul Sabatier ⁄ CNRS, Toulouse, France
Acetylcholinesterase (EC 3.1.1.7) is a serine hydrolase
that catalyzes the cleavage of acetylcholine. Structural
studies have revealed that its active site is buried in a
20 A
˚
deep gorge with a bottleneck [1]. According to
a recently developed kinetic model, substrate and
product molecules follow the same path [2]. A sub-
strate molecule first binds to the peripheral site (PAS)
at the entrance of the gorge [3] and slides down to
the acylation site (CAS), where it is hydrolyzed and
the products escape the gorge via the entrance. The
active site gorge is too narrow to allow the crossing
between a substrate molecule en route to the CAS
and a product molecule en route to the exit. Conse-

quently, at very high substrate concentrations, there
is a traffic jam preventing the exit of the reaction
product through the main entrance, resulting in inhi-
bition [4].
However, molecular dynamics experiments have pro-
vided evidence for a loop movement leading to the for-
mation of a back door suitable for product exit [5].
Locking the loop with salt or disulfide bridges [6,7]
had no significant effect on the kinetics parameters,
indicating that exit through the back door is not the
main exit route for the product. However, residual
activity upon fasciculin binding suggests that the back
door route might become the most important route
when the main entrance is blocked [8,9]. Recent kinetic
crystallography studies provide some structural insights
regarding the putative backdoor. Conformation
changes of Trp84, which belongs to the backdoor
region of Torpedo californica acetylcholinesterase, sug-
gest that this residue might behave like a revolving
door [10]. In addition, Nachon et al. [11] reported that
the back door region of the Drosophila acetylcholines-
Keywords
acetycholinesterase; back door; inhibition;
substrate; traffic
Correspondence
F. Nachon, Unite
´
d’enzymologie,
De
´

partement de Toxicologie, Centre de
Recherches du Service de Sante
´
des
Arme
´
es (CRSSA), 24 Avenue des Maquis
du Gre
´
sivaudan, 38700 La Tronche, France
Fax: +33 476636962
Tel: +33 476639765
E-mail:
(Received 18 December 2007, revised 14
March 2008, accepted 18 March 2008)
doi:10.1111/j.1742-4658.2008.06413.x
To test a product exit differing from the substrate entrance in the active
site of acetylcholinesterase (EC 3.1.1.7), we enlarged a channel located at
the bottom of the active site gorge in the Drosophila enzyme. Mutation of
Trp83 to Ala or Glu widens the channel from 5 A
˚
to 9 A
˚
. The kinetics of
substrate hydrolysis and the effect of ligands that close the main entrance
suggest that the mutations facilitate both product exit and substrate
entrance. Thus, in the wild-type, the channel is so narrow that the ‘back
door’ is used by at most 5% of the traffic, with the majority of traffic pass-
ing through the main entrance. In mutants Trp83Ala and Trp83Glu,
ligands that close the main entrance do not inhibit substrate hydrolysis

because the traffic can pass via an alternative route, presumably the
enlarged back channel.
Abbreviations
CAS, acylation site; DmAChE, Drosophila acetylcholinesterase; PAS, peripheral site.
FEBS Journal 275 (2008) 2659–2664 ª 2008 The Authors Journal compilation ª 2008 FEBS 2659
terase (DmAChE) is much less stabilized than that of
other cholinesterases, such as Torpedo californica
acetylcholinesterase. Indeed, two key residues for the
stabilization of Trp84 are not conserved in DmAChE:
Met83, which stabilizes Trp84 in the Torpedo enzyme
through sulfur-p interactions, is replaced by an isoleu-
cine; Tyr442, which hydroxyl bridges Trp84 to Trp432
and Gly80 via hydrogen bonding, is replaced by an
aspartate that is also much less bulky (Fig. 1). In the
absence of these stabilizing elements, Trp83 of
DmAChE is prone to oscillations between two alter-
nate conformations, as shown by the crystal structures
(protein databank codes 1DX4 and 1QO9). One of
these conformations results in the formation of a chan-
nel approximately 5 A
˚
in diameter, connecting the
gorge to the bulk solvent (Fig. 2A).
The present study aimed to progressively enlarge this
channel by mutating Trp83 to Tyr, Glu or Ala to test
Fig. 1. View of the back door region from the outside of DmAChE
(residues and labels in green) and Torpedo californica acetylcholin-
esterase (residues and labels in fushia). Residues are represented
by sticks. The hydrogen bonds involving the hydroxyl of Tyr442 are
indicated by a yellow dash.

A
B
Fig. 2. View of the back channel from the active site gorge of wild-
type DmAChE (A) and Trp83Ala mutant (B). The protein databank
code for wild-type DmAChE is 1DX4. Residues delimiting the hole
are represented by sticks. The solvent accessible surface is repre-
sented by a mesh.
E
K
p
S
p
E
S
p
E
K
L
ES
EA
S
p
ES
S
p
EA
EAS
S
S
S

K
p
k
2
b k
2
k
3
E
a k
3
K
p
K
L L
Choline
Choline
S
p
EAS
K
p
Acetate
Acetate
S
Scheme 1. Reaction scheme for the hydro-
lysis of acetylthiocholine by DmAChE. S,
acetylthiocholine; E, free enzyme; EA,
acetylated enzyme. All other intermediates
represent enzyme–substrate complexes and

the subscript ‘p’ denotes the substrate
bound to the peripheral anionic site.
Back channel of Drosophila acetylcholinesterase F. Nachon et al.
2660 FEBS Journal 275 (2008) 2659–2664 ª 2008 The Authors Journal compilation ª 2008 FEBS
the effect of an open back door on the kinetics for
substrate hydrolysis.
Results and Discussion
The effect of substrate concentration on acetylthiocho-
line hydrolysis for the four proteins is shown in Fig. 3.
These data were fitted using Scheme 1, which permits
the description of substrate activation and inhibition
in a manner consistent with the structural data
[4,12]. The values of the parameters for hydrolysis of
acetylthiocholine by wild-type DmAChE (Table 1) are
strongly restrained because they were deduced from
analysis of inhibition of substrate hydrolysis and accel-
eration of decarbamoylation by substrate analogue [2],
inhibition of substrate hydrolysis by reversible inhibi-
tors [13–17], hydrolysis of substrate at different tem-
peratures [18] and hydrolysis of different substrates
[19]. For the purposes of alternative substrate traffic in
and out of the active site of DmAChE, the mutation
to Tyr had no significant effect. The pS curves (i.e.
curves showing enzyme activity at different substrate
concentrations) for acetylthiocholine hydrolysis by Glu
and Ala mutants, however, were shifted to higher
concentrations of substrate and became symmetric.
Consequently, the best fits (Fig. 3) were obtained by
assuming that mutations did not affect binding of
substrate at the peripheral site (K

p
), the rate constant
for deacetylation (k
3
) and the acceleration of deacety-
lation (a). Any other assumption resulted in an unsta-
ble fitting. Consistently, it appears that substitution of
Trp83 by smaller side chains did not affect deacetyla-
tion parameters k
3
and a because the amino acid at
position 83 is too far away from the activated water
molecule during deacetylation. As expected, the main
difference is the affinity for the catalytic site K
c
(= K
p
· K
L
) because Trp83 is the main component of
substrate stabilization at the catalytic site via cation-p
interaction with the quaternary ammonium moiety of
acetylthiocholine. This is consistent with the high
apparent K
m
reported for the same mutation in human
acetylcholinesterase [20,21], although the difference
appears at different magnitudes. In addition, mutation
of this Trp to Glu or Ala decreased acylation (k
2

),
as reported for human butyrylcholinesterase [22]. Acet-
ylation may be subdivided into three steps: accommo-
dation of substrate at the CAS, chemical
transesterification and choline exit. In regard to the
effect on affinity, we can hypothesize that mutations
modify accommodation of the substrate.
Another striking difference is parameter b, which
represents the effect of substrate bound at the periph-
eral site on acylation and choline exit (Scheme 1).
Parameter b for the wild-type enzyme is estimated at
0.050 ± 0.025 (i.e. acylation step is reduced to 5%
when a substrate molecule is bound to the PAS). The
traffic of choline outside the gorge is blocked when the
PAS is occupied and choline stays inside the active
site, resulting in inhibition [9]. Parameter b is signifi-
cantly different from zero, and no combination of
parameters leading to a satisfactory fit can be obtained
if b is restrained to zero. This suggests that an alterna-
tive exit for choline may exist when the PAS is occu-
pied by a substrate molecule but would account for
approximately 5% of choline traffic. Factor b for the
Trp83Ala mutant is estimated at 1.05 (Table 1). The
acetylation step is not reduced in this mutant, suggest-
ing that choline can freely exit despite the entrance of
the gorge being occupied by a substrate molecule. This
is expected because mutation Trp83Ala enlarges the
channel by up to 9 A
˚
, thus facilitating the passage of

choline (Fig. 2B). In the case of the Glu mutation, the
b value is linked to the k
2
value and thus both cannot
be estimated independently. However, if b is set to 1
(i.e. the symmetry of the pS curve supporting it), the
Table 1. Kinetics parameters obtained for the various mutants.
Wild-type Trp83Glu Trp83Ala
k
2
(s
)1
) 52 000 ± 26 000 1818 ± 130 689 ± 30
k
3
(s
)1
) 396 ± 77 396
a
396
a
K
p
(mM) 0.175 ± 0.02 0.175
a
0.175
a
K
L
4.08 ± 2.41 38.2 ± 7.8 13.1 ± 2.4

K
LL
177 ± 33 851 ± 1.63 336 ± 63
a 3.44 ± 0.18 3.44
a
3.44
a
b 0.0498 ± 0.0247 1
a
1.05 ± 0.53
a
Parameters restrained in the simulation.
10
100 1000 10000
100 000
0
200
400
600
800
1000
W
Y
E
A
ATCh (µM)
v/Et (s
–1
)
Fig. 3. Activity of the wild-type (W) and mutated DmAChEs (Y, E,

A) at different acetylthiocholine concentrations (pS curves). Theoret-
ical curves were calculated according to the Scheme 1 specific rate
equation, using the corresponding kinetic parameters from Table 1.
F. Nachon et al. Back channel of Drosophila acetylcholinesterase
FEBS Journal 275 (2008) 2659–2664 ª 2008 The Authors Journal compilation ª 2008 FEBS 2661
fit is satisfactory. This suggests that choline can exit
freely through the back channel as in the alanine
mutant.
According to Scheme 1, inhibition by excess of
substrate does not only originate from inhibition
of choline exit (b < 1), but also from inhibition of
deacetylation following the sliding of a molecule of
substrate inside the acetylated active site and occupa-
tion of the peripheral site by a second substrate mole-
cule (complex S
p
EAS does not deacetylate in
Scheme 1). This mechanism was suggested by excess
substrate inhibition of decarbamoylation and the crys-
tal structure of the S
p
EAS obtained by soaking crystals
in a solution containing a high substrate concentration
[2,4]. This second mechanism remains active in the Glu
and Ala mutants because inhibition was observed at a
high substrate concentration (Fig. 3). We observed a
shift of the pS curve towards higher substrate concen-
trations due to the lower affinity of both the free and
acetylated mutated active sites.
If choline could leave the active site by the back

channel, we might also hypothesize that acetylcholine
enters using the same path. To test this hypothesis, we
used two inhibitors specific for the peripheral site that
bind to Trp321 close to the entrance: propidium and
aflatoxin B1. In the wild-type DmAChE, the affinity of
propidium for the peripheral site is estimated to be
80 pm, and the affinity for aflatoxin to be 3.5 lm,
when considering competition between the substrate
and inhibitor only at the PAS (Fig. 4A). However,
inhibition is completely abolished by the substitution
of Trp83 by Ala or Glu. It should be strongly empha-
sized at this point that, according to the proposed
reaction scheme (Scheme 1) enlarged by the binding of
inhibitor to the PAS, inhibition at low substrate con-
centrations should always be observed. Therefore, the
complete absence of inhibition by peripheral ligands
does not originate from changes in substrate hydrolysis
parameters (Table 1), and the simulation can readily
confirm this. The loss of inhibition following muta-
tions of Trp83 might be interpreted as a strong
decrease in affinity of ligand for the peripheral site,
resulting from a hypothetic allosteric interaction
[23,24]. However, binding to the peripheral site was
not affected by mutations, as demonstrated by changes
in fluorescence. Furthermore, considering that inhibi-
tion arises because inhibitors bound to the PAS hinder
the entrance of acetylcholine to the CAS in the wild-
type enzyme, it appears that, in the two mutants
(Trp83Ala ⁄ Glu), inhibitors that bind to the PAS did
not prevent the entrance of substrate into the active

site. At this point, a plausible explanation is that the
substrate may enter by an alternative route (i.e. the
back channel at the bottom of active site). This
hypothesis is in accordance with reported results
observed with Trp83Ala mutants: the strong decrease
of inhibition of propidium [21] and the increase of
remaining activity upon peripheral site saturation by
fasciculin [24].
Finally, minor deviations of pS curves upon binding
of inhibitors on the PAS (Fig. 4B,C), may be assigned
10 100 1000 10 000 100 000
0
200
400
600
800
1000
A
B
C
prop10 µ
M
Afl50 µ
M
ref
prop1 µ
M
prop10 µ
M
Afla10 µ

M
Afla50 µ
M
prop1 µ
M
prop10 µ
M
Afla10 µ
M
Afla50 µ
M
ref
ATCh ( M)
10 100 1000 10 000 100 000
ATCh ( M)
10 100 1000 10 000 100 000
ATCh ( M)
0
100
200
300
400
0
200
400
600
ref
v/Et (s
–1
)v/Et (s

–1
)v/Et (s
–1
)
Fig. 4. Effect of closing the entrance of the active site with ligands
(propidium or aflatoxin) on activity of wild-type DmAChE (A),
Trp83Ala (B) and Trp83Glu (C) mutants.
Back channel of Drosophila acetylcholinesterase F. Nachon et al.
2662 FEBS Journal 275 (2008) 2659–2664 ª 2008 The Authors Journal compilation ª 2008 FEBS
to an allosteric interaction between PAS and CAS
[8,21,25], to a lower efficiency of the alternative route
compared to the main entrance, or to a partial overlap
of the side chain of propidium and aflatoxin with the
active site as it may span into the gorge.
Experimental procedures
Protein production and purification
Mutations were introduced by site-directed mutagenesis
using the QuickChange XL kit following the manufac-
turer’s instructions (Stratagene, La Jolla, CA, USA). The
cDNA encoding DmAChE and mutants were expressed
with the baculovirus system [26]. We expressed a soluble
dimeric form deprived of a hydrophobic peptide at the
C-terminal and with a 3· histidine tag replacing the loop
from amino acids 103–136. This external loop is at the
opposite side of the molecule with respect to the active
site entrance and its deletion does not affect the activity
or the stability of the enzyme. Secreted acetylcholinesteras-
es were purified to homogeneity using the following steps:
ammonium sulfate precipitation, ultrafiltration with a
10 kDa cut-off membrane, affinity chromatography with

procainamide as a ligand, nitrilotriacetic acid-nickel chro-
matography and anion exchange chromatography [27].
Residue numbering follows that of the mature protein.
The concentrations of the enzymes were determined by
active site titration using high affinity irreversible inhibi-
tors [28].
Enzyme activity
Data acquisition and kinetics were performed with the sub-
strate acetylthiocholine as previously described [18]. Briefly,
the enzymatic and non-enzymatic hydrolysis of acetylthi-
ocholine by the wild-type DmAChE and its three W83
mutants was followed using Ellman’s method [29]. The
initial rate measurements were performed at acetylthiocho-
line concentrations from 2 lm to 500 mm in the absence
and presence of two ligands known to close the entrance of
the active site. We used 1 and 10 lm propidium and 10 and
50 lm aflatoxin. The activity was followed for 1 min after
the addition of acetylcholinesterase to the mixture, and the
spontaneous hydrolysis of the substrate was subtracted, if
present. Each measurement was repeated at least four
times. The experiments were carried out at 25 °Cin25mm
phosphate buffer (pH 7.0) without ionic strength compensa-
tion to avoid interference with electrostatic components of
binding and chemical steps of the reaction. Analysis of the
kinetics data were performed using gosa-fit, software that
is based on a simulated annealing algorithm (BioLog,
Toulouse, France; ). For analysis of
initial rate data in the absence of inhibitors, we used the
specific equation in Scheme 1. The effect of two ligands on
the activity of the wild-type DmAChE was evaluated by the

equation in Scheme 1 enlarged by the intermediates, repre-
senting the competition between the substrate and inhibitor
at the peripheral site [2].
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