Pressure and heat inactivation of recombinant humanacetylcholinesterase Importance of residue E202 for enzyme stability Ce´cile Cle´ry-Barraud1, Arie Ordentlich2, Haim Grosfeld2, Avigdor
Trang 1Pressure and heat inactivation of recombinant human
acetylcholinesterase
Importance of residue E202 for enzyme stability
Ce´cile Cle´ry-Barraud1, Arie Ordentlich2, Haim Grosfeld2, Avigdor Shafferman2and Patrick Masson1
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 followingexposure 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,
allowingestimation 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 phosphylatingtheir 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 modelingallowed the design of
BuChE mutants capable of degradingOPs, or slowingthe
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 addingstabilizingcomponents 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 usinghigh pressure and temperature perturbations Pressure is a convenient parameter for perturbingthe conformation and activity of enzymes [7–9] Pressure affects the structure
of folded polypeptide chains by alteringweak 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 usinggel 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: cclerybarraud@crssa.net
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)
Trang 2activity measurements followingpressure 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 9 A˚
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 bindingas the first step
in the catalytic pathway of substrate hydrolysis (Torpedo
californica AChE numberingis 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) participates in
catalytic mechanisms through a hydrogen-bond network
includingE202, Y133 and two bridgingwater 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
M A T E R I A L S A N D M E T H O D S
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 mMsodium 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 nondenaturatinggels showed that the enzyme preparations were mostly com-posed of dimeric forms (G2) of AChEs The wild-type enzyme preparation contained also traces of monomeric forms (G1) Enzyme concentrations were 2.2, 0.75, 0.60, 0.25 and 0.70 mgmL)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 25C) The specific activity of mutants were
350, 490, 30 and 580 U mg)1for E202Q, D74N, E450A and the sextuple mutant, respectively For pressure treatment, all enzyme preparations were diluted 100- to 1000-fold in
10 mM 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 (a0) at atmospheric pressure and 25C Tris buffer was chosen because its protonic activity is almost invariant with pressure up to 300 MPa [dpH/dP¼ +0.01 at 20 C due to the small ionization volume change of Tris+/Tris (DV¼ +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 kbar¼ 108Pa¼ 100 MPa) over a temperature range of 0–50C [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.
Trang 3concentrations (T%¼ 4–6.5) up to 280 MPa at 10 C in
8.26 mM Tris/glycine running buffer, pH 8.3 Enzyme
samples were diluted in the runningbuffer 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 stainingusingthe Karnovsky and Roots
method [25] with 1.7 mMATC as substrate Measurement
of enzyme and trackingdye migration distances was
performed usinga videodensitometer (Vilber-Lourmat,
Marne-la-Valle´e, France)
The mobility (m) of proteins in polyacrylamide g els is
related to the acrylamide concentration (T%) accordingto
the empirical Ferguson relationship, log m¼ log m0– KR
T%[26], where KRis the retardation coefficient, m0 is the
mobility at T%¼ 0 For globular proteins, KR is related
to the protein molecular radius R as follows K1=2R ¼
c(R + r) where c is an experimental constant and r the
radius of the polyacrylamide fiber Because R r, it follows
that KR c2(3Vh/4p)2/3, or KR,P/KR,0 (Vh,P/Vh,0)2/3
where Vhis 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 mgmL)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 mMin
10 mMTris/HCl, pH 7.5 were exposed to pressure (up to
280 MPa) for 1 h at 10C The ratio [ANS]/[AChE] (lM
concentration) was approximately 10–50 For this purpose,
Eppendorf tubes (l¼ 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 usingan SFM 25
spectrofluorimeter (Kontron) (kex¼ 358 nm) The
maxi-mal emission wavelength and maximaxi-mal 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 mM Tris buffer, pH 7.5, in an Eppendorf
tube (150 lL) Samples were exposed for 1 h at 10C under
different pressures as described above Controls were
wild-type and mutant AChEs exposed for 1 h at 10C and
atmospheric pressure (P) Because no BSA was added to
stabilize diluted AChEs, the enzyme activity progressively decreased with time at P0 Moreover, preliminary experi-ments showed that the activity of pressure-exposed enzymes continued to decrease with time after pressure release, indicatinga remnant-inactivation phenomenon as already observed for Bungarus AChE [27] and human butyrylcho-linesterase (HuBuChE) [28] Therefore, multiple rigorous controls were realized Residual activity (at) was recorded at
420 nm, exactly 5 min after decompression Activity meas-urements were performed accordingto the method of Ellman using1 mMATC as substrate in 0.1Mphosphate buffer pH 7.5 at 25C [29] The effect of pressure on activity (at) allowed to determine activation volumes of inactivation (DV„) from the slopes of plots (¶Ln at/¶P)T¼ –DV„/RT for wild-type and mutant enzymes Certain plots were not linear and we defined Ptas the transition pressure
at which a break occurred in plots of the enzyme being studied exposed to pressure for 1 h at 10C
Pressure inactivation Each AChE sample was appropri-ately diluted in 10 mMTris, pH 7.5 in an Eppendorf tube For each experiment at a given pressure and 25C, 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 25C, the residual activity was determined at atmospheric pressure and 25C, as described above Controls were enzyme samples kept at
P0 and 25C for the same periods of time (t), providing the control activity a0 at t0and the residual activity atat time t The relative activity of AChE at P0and 25C for the period of time t was the reference as (a/a0)P0,25C,t¼ 100% The relative activity of AChE submitted to pressure P, at 25C for t was (a/a0)P,25 C,t The ratio (a/a0)P,25 C,t/(a/a0)P
0,25 C,t represents the contribution of pressure to inactivation This ratio was plotted vs time to determine t1/2, 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 mMsodium phosphate buffer pH 8.0, supplemented with 0.2 mgmL)1BSA and were subjected to heat inacti-vation in 50 lL aliquots in 0.65 mL tubes (Sorenson Bioscience) Heatingtook place in a thermostatically regulated water bath, or in a PCR thermocycler (Perkin Elmer Cetus) Control unheated samples were kept at 40C until assayed For longincubation times, screw-capped, longPCR tubes (Mobitec, 100 lL) were used to prevent evaporation To calculate rate constants of inactivation, enzyme samples were heated at 55C 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 (kd) was assessed from the slope of a semilogarithmic plot, depicting residual enzyme activity as a function of time of heatingat the specific temperature T1/2 was defined as the temperature correspondingto 50% inactivation under the specified conditions For determining
T1/2, enzyme samples (50 lL) were heated in a PCR thermocycler at the indicated temperature for 10 min,
Trang 4cooled down, centrifuged (12000 g, 30 s) and assayed as
above
R E S U L T S A N D D I S C U S S I O N
Electrophoreses under pressure of wt HuAChE
and mutants
Ferguson plot analysis of gel patterns allowed us to
determine the retardation coefficient, KRat each pressure
This provided an estimation of pressure-induced changes in
hydrodynamic volume of proteins (Fig 2)
Pressure-induced dissociation of the dimeric forms (G2) of rHuAChE
was never observed up to 300 MPa, indicatingthat these
forms were native disulfide-bridged forms and not partially
proteolyzed products (disulfide-cleaved G2 forms) KR 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 swellingof the protein at around
150 MPa The mutants of rHuAChE displayed two distinct behaviors: D74N mutant exhibited a pressure dependence
of KR similar to that of wild-type enzyme, and E202Q mutant underwent the swellingtransition at higher pressures (near 200 MPa) (Fig 2A) In contrast, E450A and the sextuple mutants showed a transient increase in KR at a pressure (80 MPa) lower than for wt-rHuAChE (Fig 2B) These two mutants were less stable than wt-rHuAChE The transitory swellingwas 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-Pressure-denatured proteins have been shown to retain a compact structure with water molecules penetratingtheir 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, destabilizingthe structure and thus favoringthe 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 bindingmeasurements were performed ANS has been used for probinghydrated hydrophobic surfaces in proteins [35] and formation of molten globule-like intermediates [36,37] duringprotein 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, indicatingthat ANS bindingwas irreversible Bindingof 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 bindingof the sextuple mutant was
Fig 2 Change in the retardation coefficient, K R , with pressure for the
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.
Trang 5observed 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 bindingare in
accordance with the accepted idea that the removal of polar
residues from the hydrophobic core of globular protein has
a stabilizingeffect 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 bindingexperiments 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 bindingexperiments is not in agreement
with the high pressure sensitivity of this mutant as revealed
by electrophoresis under pressure It can be hypothesized
that bindingof 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 bindingsites may prevent water
penetra-tion in the protein core of this mutant Moreover, it has
been shown recently that ANS bindingis also favored by the
overall electric charge of proteins [38,39] As observed for
ANS bindingto BSA, bindingof 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 Bindingof multiple ANS molecules on the
protein surface could involve pressure-favored stacking
interactions and a pressure-stabilizingeffect on the protein–
ANS complex
Irreversible pressure-induced inactivation Determination of DVin„ To correlate pressure-induced conformational changes and effects of pressure on enzy-matic activity, activation volumes for pressure inactivation (DVin„) 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, allowingthe 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
DVin„ The values of calculated activation volumes for all enzymes are in Table 1 Below the break, DVin„ 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 (Pt) For the E450A and the sextuple mutants, DVin„ values are intermediate (17–
20 mL mol)1) The linear variation of Ln atvs 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 Pt Such a biphasic phenomenon has already been observed for other enzymes, for example, b-galactosidase [40] and human BuChE [28] However, the physical meaningto DVin„ is always difficult to give because DVin„ involves numerous elementary contributions, includinga 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 followingpressure treatment These meas-urements investigated the irreversible changes because no reactivation was found followingpressure release In the
Fig 3 Relative intensity of ANS fluorescence
at 469 nm in the presence of different
rHuAChEs after pressure exposure at 10 °C in
10 m M Tris buffer, pH 7.45 Symbols are as in
Fig 2 Insert: values of absolute intensity of
ANS fluorescence.
Trang 6light of electrophoresis and ANS binding experiments,
results obtained from residual activity measurements
under the same conditions (i.e 1 h exposure at 10C)
showed that the loss in activity for the wild-type
rHuAChE was concomitant with the increase in
dynamic volume and increase in solvent-exposed
hydro-phobic area at 150 MPa For the E202Q mutant, residual
activity decreased up slig htly to 180 MPa and then
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, precedingthe 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, suggestingthat duringthe 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 alongthe 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, dependingon 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 showinga Ôgrace periodÕ was observed for several mesophilic enzymes: luciferase and urease [41], and fructofuranosidase [42] It was also reported for wild-type HuBuChE duringthe 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 25C increasingwith 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
E202Q 4.62 44.07 1800
Sextuple mutant 17.9a
a No break was observed Pressure dependence of Ln (residual
activity) was linear for E450A and sextuple mutants in the pressure
range used.
Trang 7(Fig 5D) A similar activity increase with pressure was
also observed for wild-type HuBuChE at 300 MPa and
55C [44] Pressure protected the enzyme against thermal
denaturation This was interpreted as a result of the
formation of an intermediate havingan 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 ) P0,25 °C,t at 25 °C in 10 m 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.
Trang 8Thermally induced inactivation of wild-type AChE
and its mutants
Thermal inactivation was performed by heatingpurified
enzymes at 55C Heat inactivation of wild-type AChE and
selected mutants was irreversible because no spontaneous
reactivation was detected followingextended incubation of
the partially denatured AChE, at 27C (data not shown)
Table 2 depicts kdvalues of inactivation at 55C The kd
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, indicatinga t1/2 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 decreasingactivity Accordingto the
structure-function hypothesis, residues that participate in
catalysis are not optimized for stability: ÔIt should be
possible to substitute for such residues, reducingthe activity
of the protein but concomitantly increasingits stabilityÕ [45]
Thus, mutation of D74 and E202, involved in substrate
bindingand 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
Amongthe 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
in the wild-type enzyme clustered at the entrance of the 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 amongstudied mutants compared with wild-type enzyme: T1/2, the tem-perature for which the enzyme residual activity was 50%, was 62.5, 57.3 and 53C, 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 favoringhydrophobic hydration, leadingto 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-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 the active site g org e of HuAChE As the X-ray crystal 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
Trang 9gorge is thought to be lower for wild-type enzyme than for
D74N and E202Q, allowingflexibility of the gorge for
entrance and bindingof 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 throughW 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) and E197 (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]
C O N C L U S I O N
The irreversible conformational and functional alterations
of rHuAChEs were investigated using the hydrostatic
pressure approach Electrophoresis under hydrostatic
pres-sure provided evidence for prespres-sure-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„) correspondingto 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 engineeringenzyme for operatingat 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, dependingon 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 introducingadditional hydrophobic bonds on the surface or in the core, care must
be taken in interpretingresults
A C K N O W L E D G E M E N T S
We would like to thank Dr Daniel Rochu for critical readingof 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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