Báo cáo khoa học: Pressure and heat inactivation of recombinant human acetylcholinesterase Importance of residue E202 for enzyme stability pdf

Pressure and heat inactivation of recombinant human
acetylcholinesterase
Importance of residue E202 for enzyme stability
Ce
´
cile Cle
´
ry-Barraud
1
, Arie Ordentlich
2
, Haim Grosfeld
2
, Avigdor Shafferman
2
and Patrick Masson
1
1
Centre de Recherches du Service de Sante
´
des Arme
´
es, Unite
´
d’enzymologie, France;
2
Israel Institute for Biological Research,
Department of Biochemistry and Molecular Biology, Ness-Ziona, Israel
The effects of pressure on structure and activity of recom-
binant human acetylcholinesterase (rHuAChE) were inves-
tigated up to a pressure of 300 MPa using gel electrophoresis

under elevated hydrostatic pressure, fluorescence of bound
8-anilinonaphthalene-1-sulfonate (ANS) and activity meas-
urements following exposure to high pressure. Study of wild-
type enzyme and three single mutants (D74N, E202Q,
E450A) and one sextuple mutant (E84Q/E292A/D349N/
E358Q/E389Q/D390N) showed that pressure exerts a dif-
ferential action on wild-type rHuAChE and its mutants,
allowing estimation of the contribution of carboxylic amino
acid side-chains to enzyme stability. Mutation of negatively
charged residues D74 and E202 by polar side-chains
strengthened heat or pressure stability. The mutation E450A
and the sextuple mutation caused destabilization of the
enzyme to pressure. Thermal inactivation data on mutants
showed that all of them were stabilized against temperature.
In conclusion, pressure and thermal stability of mutants
provided evidence that the residue E202 is a determinant of
structural and functional stability of HuAChE.
Keywords: pressure, inactivation, protein stability, acetyl-
cholinesterase, mutants.
Acetylcholinesterase (AChE, EC 3.1.1.7) plays a central role
in the cholinergic system by rapidly hydrolyzing the
neurotransmitter acetylcholine. Organophosphorus com-
pounds (OPs), pesticides, insecticides, drugs and chemical
warfare agents (nerve gases), inhibit cholinesterases (ChEs)
by phosphylating their active-site serine. Phosphylated
ChEs can be reactivated by nucleophilic agents such as
oximes used as antidotes against organophosphate poison-
ing. Significant progresses in the treatment of poisoning by
nerve gases have been realized over the past 10 years [1,2].
However, adducts of AChE-branched OP undergo a

dealkylation, termed ÔagingÕ, which converts phosphylated
ChEs into enzymes which are impossible to reactivate. ChEs
have a therapeutic potential as exogenous scavengers for
sequestration or hydrolysis of highly toxic OPs, in particular
chemical warfare agents [3]. Biochemical data, mutagenesis,
molecular dynamics and modeling allowed the design of
BuChE mutants capable of degrading OPs, or slowing the
rate of aging. The ability to engineer ChEs resistant to aging
or able to detoxify OPs is expected to improve protection
and treatment against OP poisoning and decontamination
of harmful OP agents [4,5]. Ideally, ChE-based scavengers
should be made from a human source and have sufficient
circulatory life-time. In addition, their long-term storage
without loss of activity is suitable for economical and
operational purposes. Thus, their operational and/or con-
formational stability must be improved by chemical modi-
fication, either by adding stabilizing components or by site-
directed mutagenesis [6].
The aim of the present study was to investigate the
conformational and functional stability of AChE mutants
in order to predict whether a mutation favorable to activity
could also be favorable to stability. This is an important
issue because, in general, increasing protein conformational
stability tends to decrease the functional stability due to
decrease in flexibility. The stability of recombinant human
acetylcholinesterase (rHuAChE) was studied using high
pressure and temperature perturbations. Pressure is a
convenient parameter for perturbing the conformation
and activity of enzymes [7–9]. Pressure affects the structure
of folded polypeptide chains by altering weak interactions

responsible for stability. The extent and reversibility of
functional and structural pressure-induced changes depend
on the pressure range, the rate of compression and the
exposure time to pressure. Moderate pressure (< 300 MPa)
is a mild perturbant that does not affect the secondary
structure of proteins due to the resistance of hydrogen
bonds to pressure. Although the tertiary structure is not
significantly affected by pressure up to 300 MPa, partial
denaturation can be observed because of disruption of
hydrophobic and electrostatic interactions that are sensitive
to pressure [8].
By using gel electrophoresis under elevated pressure,
fluorescence of bound ANS after pressure exposure and
Correspondence to C. Cle
´
ry-Barraud, Centre de Recherches
du Service de Sante
´
des Arme
´
es, Unite
´
d’enzymologie,
24 Avenue des Maquis du Gre
´
sivaudan, BP 87–38702 La
Tronche ce
´
dex, France.
Fax: +33 (0)4 76636961; Tel.: +33 (0)4 76636989;

E-mail:
Abbreviations: ANS, 8-anilinonaphthalene-1-sulfonate; ATC,
acetylthiocholine iodide; ChE, cholinesterase; HuAChE, human
acetylcholinesterase; HuBuChE, human butyrylcholinesterase;
OP, organophosphorus compound.
Enzyme: acetylcholinesterase (EC 3.1.1.7).
(Received 8 April 2002, revised 8 July 2002, accepted 18 July 2002)
Eur. J. Biochem. 269, 4297–4307 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03122.x
activity measurements following pressure or heat treatment,
we investigated the structural and functional stability of
wild-type rHuAChE and four mutants (D74N, E202Q,
E450A and a sextuple mutant, E84Q/E292A/D349N/
E358Q/E389Q/D390N). These mutations were selected for
two reasons: First, to determine the contribution of
carboxylates to enzyme stability, a negatively charged
side-chain residue (E, D) was replaced by a polar (Q, N)
or a non polar (A) side-chain. Second, because of their
location in the protein: (a) the selected residues in the
sextuple mutant are located on the enzyme surface; E84,
E292, D349, and E358 encircle the gorge entrance, whereas
E389 and D390 are more distant from it; (b) E202 is lower
down in the gorge next to the active serine; (c) E450 is  9A
˚
away; and (d) D74 is located at the entrance of the active site
gorge (Fig. 1).
D74 (72) is a component of the peripheral site of
HuAChE involved in the substrate binding as the first step
in the catalytic pathway of substrate hydrolysis (Torpedo
californica AChE numbering is in parentheses when
required) [10,11]. The E202 (199)Q mutation has been

shown to affect catalysis [11,12], phosphylation [13], carb-
amylation [14] and aging of HuAChE [15,16]. The effects of
this mutation have been explained by a change in interaction
with the catalytic H447 (440) rather than by a reorganiza-
tion in the active site. Residue E450 (443)participatesin
catalytic mechanisms through a hydrogen-bond network
including E202, Y133 and two bridging water molecules
[17,18]. In addition, residues D74 and E202 have also been
shown to interact with water molecules present in the gorge
of TcAChE [19]. The other selected carboxylic residues on
the protein surface contribute to the high electrostatic
potential of the enzyme meanwhile E202 and E450 have not
been shown to contribute to the electrostatic field [20]. The
results we present on irreversible inactivation of HuAChE
by pressure and heat provide new information on the
structural and functional stability of HuAChE mutants. In
general, increasing conformational stability is known to
decrease functional stability, but we show here that a
mutation can induce stabilization of both protein structure
and activity.
MATERIALS AND METHODS
Chemicals
Acetylthiocholine iodide (ATC), 8-anilinonaphthalene-
1-sulfonate (ANS) and buffer salts were purchased from
Sigma (St Louis, MO, USA). Tris was obtained from ICN
Biomedicals (Aurora, OH, USA). Protogel (30% acryl-
amide/0.8% bis-acrylamide) was from National Diagnostics
(Atlanta, GA, USA).
Recombinant enzymes
Recombinant enzymes were expressed in human embryonic

kidney 293 cells. Mutagenesis, production in cell culture and
purification of rHuAChEs were carried out as described
previously [21]. The enzymes purified on procainamide gel
were extensively dialyzed against 50 m
M
sodium phosphate
buffer, pH 8.0. Enzymes were wild-type (wt-rHuAChE),
single mutants: E202Q, D74N, E450A, and the sextuple
mutant (E84Q/E292A/D349N/E358Q/E389Q/D390N).
Polyacrylamide electrophoresis on nondenaturating gels
showed that the enzyme preparations were mostly com-
posed of dimeric forms (G
2
) of AChEs. The wild-type
enzyme preparation contained also traces of monomeric
forms (G
1
). Enzyme concentrations were 2.2, 0.75, 0.60,
0.25 and 0.70 mg mL
)1
, respectively. The specific activity of
the wild-type enzyme was 2700 U mg
)1
with ATC as
substrate (1 U hydrolyses 1 lmol of ATC per minute at
pH 7.5 and 25 °C). The specific activity of mutants were
350, 490, 30 and 580 U mg
)1
for E202Q, D74N, E450A and
the sextuple mutant, respectively. For pressure treatment, all

enzyme preparations were diluted 100- to 1000-fold in
10 m
M
Tris/HCl buffer, pH 7.5 or 8.0. For ANS fluores-
cence and electrophoresis experiments, preparations were
diluted to the same protein concentration. For inactivation
experiments, all preparations were diluted to the same initial
activity (a
0
) at atmospheric pressure and 25 °C. Tris buffer
was chosen because its protonic activity is almost invariant
with pressure up to 300 MPa [dpH/dP ¼ +0.01 at 20 °C
due to the small ionization volume change of Tris
+
/Tris°
(DV ¼ +1 mL mol
)1
)] [22].
Electrophoreses under high pressure
We used a thermostatted high-pressure vessel, with electrical
connections as described elsewhere [23,24]. This vessel,
suited for microdisc electrophoresis, can operate up to a
pressure of 300 MPa (1 kbar ¼ 10
8
Pa ¼ 100 MPa) over a
temperature range of 0–50 °C [23]. Silicon oil (DC200,
100 cst) was the pressure vector because it is inert, non
compressible and non conducting. High pressure electro-
phoreses were performed in polyacrylamide capillary gel
rods (/ ¼ 1 · l ¼ 75 mm) of different acrylamide

Fig. 1. Schematic view of the 3D folding of modeled rHuAChE mono-
mer in an orientation (gorge entrance at the top) showing the distribution
of mutated carboxylic amino acids. D74, E202 and E450 are in the
active site gorge and E84/E292/D349/E358/E389/D390 are around the
gorge entrance.
4298 C. Cle
´
ry-Barraud et al. (Eur. J. Biochem. 269) Ó FEBS 2002
concentrations (T
%
¼ 4–6.5) up to 280 MPa at 10 °Cin
8.26 m
M
Tris/glycine running buffer, pH 8.3. Enzyme
samples were diluted in the running buffer and loaded onto
the gels. Six gels were simultaneously submitted to the
desired pressure for thermodynamic equilibration for a
period of 10 min. Then, electrophoreses were carried out for
15 min at a constant intensity (0.3 mA gel
)1
) under this
pressure. Electrophoreses were performed in triplicate for
each enzyme under given pressure. The enzyme bands were
detected by activity staining using the Karnovsky and Roots
method [25] with 1.7 m
M
ATC as substrate. Measurement
of enzyme and tracking dye migration distances was
performed using a videodensitometer (Vilber-Lourmat,
Marne-la-Valle

´
e, France).
The mobility (m)ofproteinsinpolyacrylamidegelsis
related to the acrylamide concentration (T
%
) according to
the empirical Ferguson relationship, log m ¼ log m
0
– K
R
T
%
[26], where K
R
is the retardation coefficient, m
0
is the
mobility at T
%
¼ 0. For globular proteins, K
R
is related
to the protein molecular radius R as follows K
1=2
R
¼
c(R + r) where c is an experimental constant and r the
radius of the polyacrylamide fiber. Because R  r, it follows
that K
R

 c
2
(3V
h
/4p)
2/3
,orK
R,P
/K
R,0
 (V
h,P
/V
h,0
)
2/3
where V
h
is the hydrodynamic volume of the protein and
subscripts P and 0 refer to values at pressure P and at
atmospheric pressure, respectively [23].
ANS fluorescence after pressure exposure of AChE
in the presence of ANS
The different rHuAChEs (0.077, 0.041, 0.049, 0.017 and
0.05 mg mL
)1
for wild-type rHuAChE, D74N, E202Q,
E450A and sextuple mutant, respectively) in the presence
and absence of ANS at a final concentration of 0.11 m
M

in
10 m
M
Tris/HCl, pH 7.5 were exposed to pressure (up to
280 MPa) for 1 h at 10 °C. The ratio [ANS]/[AChE] (l
M
concentration) was approximately 10–50. For this purpose,
Eppendorf tubes (l ¼ 20 · / ¼ 4 mm) filled with the
mixture of ANS and enzyme solutions were sealed with a
latex membrane maintained with an O-ring. All tubes were
immersed in the cell compartment of the temperature-
controlled high-pressure vessel. After pressure release,
fluorescence emission spectra of free and bound ANS were
recorded between 400 and 550 nm using an SFM 25
spectrofluorimeter (Kontron) (k
ex
¼ 358 nm). The maxi-
mal emission wavelength and maximal fluorescence inten-
sity were determined after each pressure exposure to obtain
information on irreversible pressure-induced structural
changes. Spectra of bound ANS were corrected by
subtraction of the fluorescence spectra of free ANS in the
buffer at each pressure. No significant change in free ANS
fluorescence was observed after pressure release. For each
pressure, at least two spectra were recorded and averaged.
Irreversible pressure-induced inactivation
Determination of activation volume of inactivation
(V
„
in

) Each enzyme was diluted in an appropriate
manner in 10 m
M
Tris buffer, pH 7.5, in an Eppendorf
tube (150 lL). Samples were exposed for 1 h at 10 °C under
different pressures as described above. Controls were wild-
type and mutant AChEs exposed for 1 h at 10 °Cand
atmospheric pressure (P
0
). Because no BSA was added to
stabilize diluted AChEs, the enzyme activity progressively
decreased with time at P
0
. Moreover, preliminary experi-
ments showed that the activity of pressure-exposed enzymes
continued to decrease with time after pressure release,
indicating a remnant-inactivation phenomenon as already
observed for Bungarus AChE [27] and human butyrylcho-
linesterase (HuBuChE) [28]. Therefore, multiple rigorous
controls were realized. Residual activity (a
t
) was recorded at
420 nm, exactly 5 min after decompression. Activity meas-
urements were performed according to the method of
Ellman using 1 m
M
ATC as substrate in 0.1
M
phosphate
buffer pH 7.5 at 25 °C [29]. The effect of pressure on

activity (a
t
) allowed to determine activation volumes of
inactivation (DV
„
) from the slopes of plots (¶Ln a
t
/¶P)
T
¼
–DV
„
/RT for wild-type and mutant enzymes. Certain plots
were not linear and we defined P
t
as the transition pressure
atwhichabreakoccurredinplotsoftheenzymebeing
studied exposed to pressure for 1 h at 10 °C.
Pressure inactivation Each AChE sample was appropri-
ately diluted in 10 m
M
Tris, pH 7.5 in an Eppendorf tube.
For each experiment at a given pressure and 25 °C,
enzymes samples were subjected to hydrostatic pressure
up to 300 MPa for different periods of time ranging from
5 min to 3 h. Then, exactly 5 min after pressure release
and thermal equilibration at 25 °C, the residual activity
was determined at atmospheric pressure and 25 °C, as
described above. Controls were enzyme samples kept at
P

0
and 25 °C for the same periods of time (t), providing
the control activity a
0
at t
0
and the residual activity a
t
at
time t. The relative activity of AChE at P
0
and 25 °Cfor
the period of time t was the reference as (a/a
0
)
P
0
,25 °C,t
¼
100%. The relative activity of AChE submitted to
pressure P,at25°Cfort was (a/a
0
)
P,25 °C,t
.Theratio
(a/a
0
)
P,25 °C,t/
(a/a

0
)
P
0
,
25 °C,t
represents the contribution of
pressure to inactivation. This ratio was plotted vs. time to
determine t
1/2
, the time at which the enzyme retained 50%
of its initial activity.
Thermal inactivation
Purified AChEs were diluted to approximately 0.3 U mL
)1
in 50 m
M
sodium phosphate buffer pH 8.0, supplemented
with 0.2 mg mL
)1
BSA and were subjected to heat inacti-
vation in 50 lL aliquots in 0.65 mL tubes (Sorenson
Bioscience). Heating took place in a thermostatically
regulated water bath, or in a PCR thermocycler (Perkin
Elmer Cetus). Control unheated samples were kept at 40 °C
until assayed. For long incubation times, screw-capped,
long PCR tubes (Mobitec, 100 lL) were used to prevent
evaporation. To calculate rate constants of inactivation,
enzyme samples were heated at 55 °C for various lengths of
time, cooled quickly in iced water to stop inactivation,

centrifuged for 2 min at 10 000 g and assayed for residual
activity. In each experiment, the five enzyme preparations
were heat-treated simultaneously. The first-order denatur-
ation rate constant (k
d
) was assessed from the slope of a
semilogarithmic plot, depicting residual enzyme activity as a
function of time of heating at the specific temperature. T
1/2
was defined as the temperature corresponding to 50%
inactivation under the specified conditions. For determining
T
1/2
, enzyme samples (50 lL) were heated in a PCR
thermocycler at the indicated temperature for 10 min,
Ó FEBS 2002 Inactivation of human acetylcholinesterase (Eur. J. Biochem. 269) 4299
cooled down, centrifuged (12000 g, 30 s) and assayed as
above.
RESULTS AND DISCUSSION
Electrophoreses under pressure of wt HuAChE
and mutants
Ferguson plot analysis of gel patterns allowed us to
determine the retardation coefficient, K
R
at each pressure.
This provided an estimation of pressure-induced changes in
hydrodynamic volume of proteins (Fig. 2). Pressure-
induced dissociation of the dimeric forms (G
2
)ofrHuAChE

was never observed up to 300 MPa, indicating that these
forms were native disulfide-bridged forms and not partially
proteolyzed products (disulfide-cleaved G
2
forms). K
R
of
the dimeric wild-type rHuAChE was almost constant up to
120 MPa; it increased up to 150 MPa and then dropped
(Fig. 2). A similar behavior was previously observed
for human BuChE [24]. This was interpreted as a
pressure-induced swelling of the protein at around
150 MPa. The mutants of rHuAChE displayed two distinct
behaviors: D74N mutant exhibited a pressure dependence
of K
R
similar to that of wild-type enzyme, and E202Q
mutant underwent the swelling transition at higher pressures
(near 200 MPa) (Fig. 2A). In contrast, E450A and the
sextuple mutants showed a transient increase in K
R
at a
pressure (80 MPa) lower than for wt-rHuAChE (Fig. 2B).
These two mutants were less stable than wt-rHuAChE. The
transitory swelling was thought to be due to penetration of
water into the protein core and at the subunit interface. This
suggests the occurrence of a pressure-induced stable inter-
mediate state for wild-type and mutant enzymes at different
pressures ranging between 80 and 200 MPa. These results
are in agreement with experimental findings reported for

protein molten-globule transitions, i.e. an increase in the
hydrodynamic radius of proteins upon denaturation [24,30],
and an increase in the hydrogen-exchange rates as seen for
lysozyme and RNase A with pressure [31]. Pressure-
denatured proteins unlike heat-denatured proteins have
been shown to retain a compact structure with water
molecules penetrating their core as probed by NMR
experiments of hydrogen exchange [32]. In this context, it
can be suggested that the replacement of E by A may have
created a 27-A
˚
cavity [33] in AChE, destabilizing the
structure and thus favoring the penetration of water in the
E450A mutant at a pressure lower than for pressure-favored
penetration of water in wild-type enzyme. For the sextuple
mutant, electrophoresis under pressure data indicated that
the removal of several carboxyl groups at the protein
surface destabilizes the protein. These charged residues may
be involved in salt bridges that stabilize the folded protein.
Indeed, surface salt bridges contribute to protein stability,
but Takano et al. have shown that contribution of salt
bridges to protein stability is variable, depending on their
structural characteristic and their location on the surface
[34].
ANS binding
To investigate further the description of the mechanism of
the pressure denaturation process of rHuAChE, ANS
binding measurements were performed. ANS has been used
for probing hydrated hydrophobic surfaces in proteins [35]
and formation of molten globule-like intermediates [36,37]

during protein denaturation processes. Fluorescence of
bound ANS was progressively enhanced, indicating that
ANS progressively bound to enzyme. Figure 3 shows the
relative fluorescence intensity at 469 nm of ANS bound to
wild-type and mutant rHuAChEs as a function of pressure.
Fluorescence intensity spectra monitored vs. time after
pressure release (from 5 min to 18 h) did not return to the
initial spectra, indicating that ANS binding was irreversible.
Binding of ANS to wt-rHuAChE transiently increased with
pressures up to 100 MPa, then dropped, and then increased
considerably again beyond 125 MPa. D74N and E450A
mutants showed a slight enhancement of ANS binding from
125 MPa and E202Q from 200 MPa compared with wild-
type enzyme. This suggests that pressure denaturation (i.e.
appearance of newly solvent-exposed hydrophobic residues)
of these mutants was less extended than for wild-type
enzyme over the same pressure range. Otherwise, no
increase in ANS binding of the sextuple mutant was
Fig. 2. Change in the retardation coefficient, K
R
,withpressureforthe
dimeric form of rHuAChEs and mutants in 8.26 m
M
Tris buffer/0.1
M
glycine, pH 8.3, at 10 °C. (A) Wild-type rHuAChE (d); D74N (m);
E202Q (j). (B) Wild-type rHuAChE (d); E450A (.); sextuple mutant
(·). Error bars indicate standard deviations for 3–5 independent
measurements.
4300 C. Cle

´
ry-Barraud et al. (Eur. J. Biochem. 269) Ó FEBS 2002
observed in the relevant pressure range. Moreover, the
sextuple mutant showed the strongest affinity for the probe
at atmospheric pressure (Fig. 3, insert), indicating that its
native state was different from the initial state of other
enzymes, and that this mutant has solvent-accessible
hydrophobic patches in its native conformation. Except
for the sextuple mutant, results on ANS binding are in
accordance with the accepted idea that the removal of polar
residues from the hydrophobic core of globular protein has
a stabilizing effect. Thus, the E450A and the E202Q
mutants are more stable than the D74N mutant and the
wild type. The pressure insensitivity of the E202Q mutant as
seen from ANS binding experiments and electrophoresis
may be explained by a decreased flexibility in the active site
gorge, preventing this mutant from pressure denaturation.
Replacement of E by Q (or D by N) maintains potential
hydrogen-bond interactions but causes disruption of any
ionic interactions. Thus, creation of additional hydrogen
bonds that are known to be pressure insensitive could be at
the origin of the marked stability of E202Q mutant under
pressure. The pressure insensitivity of the sextuple mutant as
seen from ANS binding experiments is not in agreement
with the high pressure sensitivity of this mutant as revealed
by electrophoresis under pressure. It can be hypothesized
that binding of the dye to protein surface sites protects
protein against pressure. This is supported by a report on
the protective effect of ANS against thermal and acid pH
shocks [38]. Thus, multiple interactions between ANS and

solvent-exposed binding sites may prevent water penetra-
tion in the protein core of this mutant. Moreover, it has
been shown recently that ANS binding is also favored by the
overall electric charge of proteins [38,39]. As observed for
ANS binding to BSA, binding of ANS is thought to induce
a conformational tightening of the protein by the interplay
of ionic and hydrophobic characters of both protein and
ANS molecules. Binding of multiple ANS molecules on the
protein surface could involve pressure-favored stacking
interactions and a pressure-stabilizing effect on the protein–
ANS complex.
Irreversible pressure-induced inactivation
Determination of DV
in
„
To correlate pressure-induced
conformational changes and effects of pressure on enzy-
matic activity, activation volumes for pressure inactivation
(DV
in
„
) of wild-type and mutant enzymes were determined
(Fig. 4). Two distinct types of behavior were observed. For
wild-type AChE, D74N and E202Q mutants, plots were
biphasic, allowing the estimation of two activation volumes,
on both sides of the break. The E450A and the sextuple
mutant showed a linear pressure dependence of the
inactivation process characterized by a single positive
DV
in

„
. The values of calculated activation volumes for all
enzymes are in Table 1. Below the break, DV
in
„
are small
(3–11 mL mol
)1
) for the E202Q and D74N mutants and
wild-type enzyme but much larger (30–45 mL mol
)1
)
beyond the pressure transition (P
t
). For the E450A and
the sextuple mutants, DV
in
„
values are intermediate (17–
20 mL mol
)1
). The linear variation of Ln a
t
vs. P indicates
that neither pressure-induced conformational change nor
compressibility change occurred for both E450A and
sextuple mutants in the relevant pressure range. However,
biphasic plots for wild-type enzyme, D74N and E202Q
mutants suggest an effect of pressure on the enzyme
structure. This effect is probably due to the formation of a

second active conformation at pressures higher than P
t
.
Such a biphasic phenomenon has already been observed for
other enzymes, for example, b-galactosidase [40] and human
BuChE [28]. However, the physical meaning to DV
in
„
is
always difficult to give because DV
in
„
involves numerous
elementary contributions, including a configuration term
(changes in polypeptide chain conformation), an intramo-
lecular term (changes in short- and long-range interactions)
and hydration changes.
In this study, three different properties (i.e. electro-
phoresis mobility, ANS fluorescence and enzyme activity)
were measured following pressure treatment. These meas-
urements investigated the irreversible changes because no
reactivation was found following pressure release. In the
Fig. 3. Relative intensity of ANS fluorescence
at 469 nm in the presence of different
rHuAChEs after pressure exposure at 10 °Cin
10 m
M
Tris buffer, pH 7.45. Symbols are as in
Fig. 2. Insert: values of absolute intensity of
ANS fluorescence.

Ó FEBS 2002 Inactivation of human acetylcholinesterase (Eur. J. Biochem. 269) 4301
light of electrophoresis and ANS binding experiments,
results obtained from residual activity measurements
under the same conditions (i.e. 1 h exposure at 10 °C)
showed that the loss in activity for the wild-type
rHuAChE was concomitant with the increase in hydro-
dynamic volume and increase in solvent-exposed hydro-
phobic area at 150 MPa. For the E202Q mutant, residual
activitydecreasedupslightlyto180MPaandthen
decreased further while the hydrodynamic volume
increased and more hydrophobic patches became exposed
to the solvent. The activity of the D74N mutant slightly
decreased up to 100 MPa and then dropped while the
hydrodynamic volume increased, preceding the exposure
of hydrophobic areas. For the E450A and sextuple
mutants, the hydrodynamic volume increased at a lower
pressure (80 MPa), but the residual activity decreased
linearly with pressure and, as for other rHuAChEs, the
hydrophobic areas became more exposed to the solvent
beyond 125 MPa (except for the sextuple mutant).
Pressure-induced changes in ANS binding, electrophoresis
mobility and residual activity did not appear at the same
pressure, suggesting that during the course of pressure
denaturation, several intermediates were generated. One of
them was characterized: its hydrodynamic volume was
increased compared with that of native enzyme, some of
its hydrophobic residues were newly exposed to solvent,
and it remained active.
Pressure inactivation of HuAChE and its mutants To test
the hypothesis of several intermediates along the pressure

denaturation process, pressure inactivation of wild-type
rHuAChE and its mutants was carried out. Figure 5 shows
the effect of the pressurization duration on the remaining
activity as determined 5 min after pressure release. No
hysteresis was seen for the residual activity of the various
enzymes exposed to pressure. The decompression speed was
about 20 s per 100 MPa so that the pressure release took
place in less than 1 min at 300 MPa. For all rHuAChEs, the
time course of inactivation was biphasic and complex. Two
distinct patterns were observed, depending on the enzyme
type. In the first pattern, a fast inactivation phase was
followed by a slower process (concave curves). This
behavior was observed for the sextuple mutant (Fig. 5A)
and for other enzymes at pressures above 200 MPa. In the
second pattern, after an initial phase with no change in
activity, there was either enzyme inactivation between 80
and 100 MPa for wild type (Fig. 5B), at 100 MPa for
E450A (Fig. 5C), or enzyme activation at 100 MPa for
E202Q (Fig. 5D). The initial phase was termed the Ôgrace
periodÕ. Thermal inactivation showing a Ôgrace periodÕ was
observed for several mesophilic enzymes: luciferase and
urease [41], and fructofuranosidase [42]. It was also reported
for wild-type HuBuChE during the first 5 min of ultrasound
inactivation kinetics [43] and after pressure/temperature
exposure [28].
E202Q was the most pressure resistant mutant. This
mutant showed pressure-induced activation at 100 MPa
and 25 °C increasing with the exposure time to pressure
Fig. 4. Pressure dependence of percentage of
residual activity of rHuAChE vs. pressure at

10 °C after 1 h of pressure exposure in 10 m
M
Tris buffer (pH 8.0). Symbols are as in Fig. 2.
Table 1. Values of activation volumes (DV
in
„
) of inactivation for wild-
type and mutant rHuAChEs calculated from the slope of Ln (% residual
activity) vs. pressure. The transition pressure (P
t
) is the pressure at
which a break occurs in the plot.
Enzyme
DV
in
„
(mL mol
)1
)
P < P
t
P > P
t
P
t
(bar)
Wild type 11.3 31.9 1500
D74N 3.73 36.7 1000
E202Q 4.62 44.07 1800
E450A 19.7

a
––
Sextuple mutant 17.9
a
a
No break was observed. Pressure dependence of Ln (residual
activity) was linear for E450A and sextuple mutants in the pressure
range used.
4302 C. Cle
´
ry-Barraud et al. (Eur. J. Biochem. 269) Ó FEBS 2002
(Fig. 5D). A similar activity increase with pressure was
also observed for wild-type HuBuChE at 300 MPa and
55 °C [44]. Pressure protected the enzyme against thermal
denaturation. This was interpreted as a result of the
formation of an intermediate having an activity higher
than that of the native enzyme. Present results on
HuAChE mutants also suggest that by modulating the
temperature and pressure parameters, we can induce
formation of intermediates more active than the native
enzymes.
Fig. 5. Ratio of residual activity of rHuAChE (a/a
0
)
P,25 °C,t
/(a/a
0
)
P
0

,25 °C,t
at 25 °Cin10m
M
Tris buffer, pH 7.5, as a function of exposure time under
different pressures (P). Symbols represent pressures as follows: 500 bar (n); 800 bar (s); 1000 bar (d); 1500 bar (,); 2000 bar (m); 3000 bar (j). P
0
refers to atmospheric pressure. (A) Sextuple mutant; (B) wild type; (C) E450A; (D) E202Q; and (E) D74N.
Ó FEBS 2002 Inactivation of human acetylcholinesterase (Eur. J. Biochem. 269) 4303
Thermally induced inactivation of wild-type AChE
and its mutants
Thermal inactivation was performed by heating purified
enzymes at 55 °C. Heat inactivation of wild-type AChE and
selected mutants was irreversible because no spontaneous
reactivation was detected following extended incubation of
the partially denatured AChE, at 27 °C (data not shown).
Table 2 depicts k
d
values of inactivation at 55 °C. The k
d
value of the E450A mutant is more than 10-fold lower than
that of wild-type AChE, and that of E202Q is more than
500-fold lower than wild-type, indicating a t
1/2
of 72 h.
However, replacement of six carboxylic residues vicinal to
the rim of the active site center gorge induced a heat stability
not so different from that of the single point mutated
enzyme D74N, which is slightly more stable than the wild
type (Table 2). Our results suggest that the E202Q mutant
was the most stable enzyme after temperature treatment and

allow us to rank rHuAChE mutants and wild-type enzyme
in term of thermostability: E202Q  E450A > D74N >
sextuple mutant > wild-type. Enzyme thermostability is
often explained by high rigidity of molecular structure
accompanied by decreasing activity. According to the
structure-function hypothesis, residues that participate in
catalysis are not optimized for stability: ÔIt should be
possible to substitute for such residues, reducing the activity
of the protein but concomitantly increasing its stabilityÕ [45].
Thus, mutation of D74 and E202, involved in substrate
binding and catalysis [10–12], determined a better heat or
pressure stability when their negatively charged side-chains
were replaced by polar side-chains.
Comparison of pressure and thermal stability
High hydrostatic pressure and temperature are known to
exert antagonistic effects on weak interactions. It is accepted
that formation of hydrophobic contacts and electrostatic
interactions that proceed with a positive variation of volume
are disfavored by pressure [8,46] but favored by temperature
increase. The formation of hydrogen bonds is rather
pressure insensitive but disfavored by temperature increase
[47]. Stacking of aromatic rings and charge-transfer inter-
actions are pressure-stabilized [8]. Thus, the stability of
proteins is expected to be affected in different ways by these
two variables [48]. This was verified for the E450A and
sextuple mutants which were destabilized by pressure but
stabilized by temperature, compared with wild-type enzyme.
Among the rHuAChEs we analyzed, the sextuple mutant
exhibited the weakest stability to pressure but a temperature
stability quite similar to that of wild-type enzyme. This

observation suggests again that negatively charged residues
inthewild-typeenzymeclusteredattheentranceofthe
gorge are involved in stabilizing interactions. As already
shown for other proteins, electrostatic interactions play a
major role in stability [49–51]. Moreover, as for halophilic
and thermophilic proteins, the apparent requirement for so
many acidic groups on the surface of AChE could be
rationalized on the basis of their high water-binding ability
compared with other amino acid side-chains [52]. Because a
few of the numerous negative charges located at the rim of
the gorge did not participate to the entrance of substrate or
ligand in the gorge [20], we tentatively suggest that this
cluster of charges could be involved in function and
stability, relevant to the AChE location in cholinergic
synapses [53].
It appeared that in addition to its higher pressure
conformational stability and activity, the E202Q mutant
was also the most temperature resistant among studied
mutants compared with wild-type enzyme: T
1/2
,thetem-
perature for which the enzyme residual activity was 50%,
was 62.5, 57.3 and 53 °C, for E202Q, E450A and wild-type
AChE, respectively (results not shown). At present, we can
only speculate on the structural cause of pressure and
temperature stability of the E202Q mutant. Substitution of
E by Q may preserve potential hydrogen-bond interactions
but causes disruption of ionic interactions.
Hei and Clark have suggested that hydrophobic interac-
tions responsible for the stabilization of several thermosta-

ble enzymes also contribute to pressure stabilization of
enzymes from thermophilic organisms [54]. Temperature
and pressure would not affect hydrophobic interactions in
the protein core but only those at the protein surface by
favoring hydrophobic hydration, leading to a greater
protein rigidity but allowing a flexibility in the active site
gorge for enzyme activity. This interpretation may explain
the behavior of the E450A mutant (where a charged side-
chain was substituted by a hydrophobic side-chain). This
residue is in the protein core down the active site gorge and,
thus, exposed to hydrophobic hydration. This suggests that
protein rigidity in the active site gorge involves a decrease in
activity. To determine the real structural consequences of
these substitutions, the X-ray crystal structures of these
mutants should be determined.
Moreover, certain carboxylic residues in the active site
gorge (D74 at the rim, E202 and E450 at the bottom of the
gorge, respectively) were shown to be involved in the
pressure sensitivity of HuAChE as for HuBuChE and its
D70G (D72) and E197D (E199) mutants [28,55]. The
intermediate states observed are probably due to hydration
change and compressibility change associated with a
conformational change in the gorge. Wild-type enzyme,
D74N and E202Q mutants were found to be more stable
than E450A and pressure denaturation showed that E202Q
is the most stable enzyme. These results can be interpreted in
terms of hydration change of carboxylic residues present in
theactivesitegorgeofHuAChE.AstheX-raycrystal
structures of TcAChE [19] and HuAChE [56] revealed, the
conserved position of water molecules in the active site

gorges of TcAChE and HuAChE is evidence for the
importance of water in the structure of AChE. The structure
and number of water molecules in the gorge and bound to
the enzyme have been shown to play a substantial role in the
conformational stability and reactions catalyzed by cho-
linesterases [55]. The coordination of water molecules in the
Table 2. The first-order rate constant (k
d
) of heat inactivation at 55 °C
of HuAChE and selected mutants.
HuAChE k
d
(· 10
2
min
)1
)
Wild type 10.0 ± 1.2
E84Q/E292A/D349N/E358Q/E389Q/D390N 5.2 ± 1.6
D74N 4.0 ± 0.6
E450A 0.79 ± 0.15
E202Q 0.016 ± 0.05
4304 C. Cle
´
ry-Barraud et al. (Eur. J. Biochem. 269) Ó FEBS 2002
gorge is thought to be lower for wild-type enzyme than for
D74N and E202Q, allowing flexibility of the gorge for
entrance and binding of substrates or inhibitors and exit of
reaction products. The carboxylic groups of E202 and E450,
the hydroxyl group of Y133 and water molecules are

involved in a hydrogen-bond network, as described by
Ordentlich et al. [17]. This network is thought to have two
roles: (a) It participates in structural stabilization of
transition states as replacement of E202 or E450 affects
the catalysis of both charged and noncharged substrates
[55,57]. (b) It maintains the conformation of E202 for
optimal interactions with the active site actors. However, the
X-ray structure of the E202Q mutant complexed with
fasciculin II showed the existence of the hydrogen-bond
network [Y133–water–E202–water–E450] as in the complex
with the wild type [53]. The E202Q mutation did not disrupt
the network at the bottom of the gorge, but the active site
area in this mutant might be less solvated than that of the
wild-type enzyme. The effect of D74N mutation on
hydration of the gorge could be indirect, resulting from a
change in the conformation of the gorge through W loop
motion [58,59]. Results we have presented here are in good
agreement with those previously obtained on the effects of
osmotic and hydrostatic pressure on the aging of phospho-
rylated BuChE. This double approach allowed us to probe
the participation of water in the mechanism of aging. It was
shown that residues D70 (72)andE197(199) affect the
water-stabilized transition state of dealkylation [55]. More-
over, residue D70 was found to be involved in conforma-
tional stability of BuChE and in activation by excess
substrate [60,61].
CONCLUSION
The irreversible conformational and functional alterations
of rHuAChEs were investigated using the hydrostatic
pressure approach. Electrophoresis under hydrostatic pres-

sure provided evidence for pressure-induced hydrodynamic
volume change of these enzymes. Fluorescence of ANS
bound to wild-type and mutant rHuAChEs indicated
solvent exposure changes of hydrophobic residues during
the pressure denaturation process. Measurement of the
residual activity of enzymes after pressure exposure allowed
to calculate activation volumes (DV
„
) corresponding to the
irreversible enzyme inactivation. The DV
„
values were
correlated to the observed conformational changes. All
the results showed that pressure exerts a differential action
on wild-type rHuAChE and its mutants: e.g. the E202Q
mutant showed resistance to high pressure while E450A and
the sextuple mutant are sensitive. The results reported show
that pressure induces a number of intermediates between the
folded and unfolded enzyme states. This conclusion comes
from the observation that pressure-induced changes in
different properties (ANS binding, electrophoresis mobility,
activity) do not superimpose. Moreover, we found that
certain denaturation intermediates are more active than the
native states. The existence of these stable intermediate
states accounts for nonlinearity of inactivation kinetics.
Analysis of the pressure effects on rHuAChE also showed
that engineering enzyme for operating at high pressure can
increase both functional and structural stability, as for the
E202Q mutant. A mutated enzyme that is thermodynam-
ically more stable and more active than the wild-type

enzyme is of potential interest for the design of new ChE-
based OP scavengers which are more stable upon storage.
Results also showed that the effects of pressure and
temperature can lead to opposite conclusions about the
role of elementary interactions in the conformational or
functional protein stability, depending on both the type and
location of mutation and/or the experimental technique.
This means that to investigate whether the stability of
proteins can be further enhanced by introducing additional
hydrophobic bonds on the surface or in the core, care must
be taken in interpreting results.
ACKNOWLEDGEMENTS
We would like to thank Dr Daniel Rochu for critical reading of the
manuscript. We are grateful to J L. Saldana for helping us with the
high pressure apparatus assembly and maintenance. This work was
supported by DGA/DSP/STTC, grant number DRET 96/12 to PM
and AS.
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Ó FEBS 2002 Inactivation of human acetylcholinesterase (Eur. J. Biochem. 269) 4307