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: 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) 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. 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Masson, P (1999) Differential effect of pressure and temperature on the catalytic behaviour of wild-type human butyrylcholinesterase and its D70G mutant Eur J Biochem 264, 327–335 61 Levitsky, V., Xie, W., Froment, M.-T., Lockridge, O & Masson, P (1999) Polyol-induced activation by excess substrate of the D70G butyrylcholinesterase mutant Biochim Biophys Acta 1429, 422–430 . Pressure and heat inactivation of recombinant human acetylcholinesterase Importance of residue E202 for enzyme stability Ce ´ cile. conclusion, pressure and thermal stability of mutants provided evidence that the residue E202 is a determinant of structural and functional stability of HuAChE. Keywords: