Mechanisms of cholinesterase inhibition by inorganic mercury Manuela F. Frasco 1,2 , Jacques-Philippe Colletier 3 , Martin Weik 3 ,Fe ´ lix Carvalho 4 , Lu ´ cia Guilhermino 1,2 , Jure Stojan 5 and Didier Fournier 6 1 ICBAS, Instituto de Cie ˆ ncias Biome ´ dicas de Abel Salazar, Universidade do Porto, Portugal 2 CIMAR-LA ⁄ CIIMAR, Universidade do Porto, Portugal 3 IBS-UMR 5075, CEA-CNRS-UJF, Laboratoire de Biophysique Mole ´ culaire, Grenoble, France 4 REQUIMTE, Servic¸o de Toxicologia da Faculdade de Farma ´ cia da Universidade do Porto, Portugal 5 Institute of Biochemistry, University of Ljubljana, Slovenia 6 IPBS-UMR 5089, CNRS-UPS, Groupe de Biotechnologie des Prote ´ ines, Toulouse, France Human activities have continuously contaminated the environment with mercury, which has been used for centuries in agriculture, industry, and medicine [1]. Nowadays, inorganic mercury is used in, for example, thermometers, batteries, and fluorescent light-bulbs. In addition, large quantities of metallic mercury are employed in the fabrication of electrodes for the elec- trolytic production of chlorine and sodium hydroxide from salt, as well as in gold mining [2,3]. Although it presents unique properties that make it useful for many human purposes, mercury has no role in life processes and is highly toxic. Nephrotoxicity [4] and genotoxicity [5] have been demonstrated. Other adverse effects occur in neural tissues, where the targeting of enzymes and receptors involved in nerve impulse trans- mission is probably involved [6], as well as in the Keywords aggregation; cholinesterase; inhibition; mercury; metals Correspondence D. Fournier, IPBS-UMR 5089, 205 Route de Narbonne, F-31077 Toulouse, France Fax: +33 5 61 17 59 94 Tel: +33 5 61 55 54 45 E-mail: fournier@ipbs.fr Database The coordinates and structure factor ampli- tudes of the complex structure of human butyrylcholinesterase with HgCl 2 have been deposited in the Protein Data Bank under accession code 2J4C (Received 18 December 2006, revised 1 February 2007, accepted 7 February 2007) doi:10.1111/j.1742-4658.2007.05732.x The poorly known mechanism of inhibition of cholinesterases by inorganic mercury (HgCl 2 ) has been studied with a view to using these enzymes as biomarkers or as biological components of biosensors to survey polluted areas. The inhibition of a variety of cholinesterases by HgCl 2 was investi- gated by kinetic studies, X-ray crystallography, and dynamic light scatter- ing. Our results show that when a free sensitive sulfhydryl group is present in the enzyme, as in Torpedo californica acetylcholinesterase, inhibition is irreversible and follows pseudo-first-order kinetics that are completed within 1 h in the micromolar range. When the free sulfhydryl group is not sensitive to mercury (Drosophila melanogaster acetylcholinesterase and human butyrylcholinesterase) or is otherwise absent (Electrophorus electri- cus acetylcholinesterase), then inhibition occurs in the millimolar range. Inhibition follows a slow binding model, with successive binding of two mercury ions to the enzyme surface. Binding of mercury ions has several consequences: reversible inhibition, enzyme denaturation, and protein aggregation, protecting the enzyme from denaturation. Mercury-induced inactivation of cholinesterases is thus a rather complex process. Our results indicate that among the various cholinesterases that we have studied, only Torpedo californica acetylcholinesterase is suitable for mercury detection using biosensors, and that a careful study of cholinesterase inhibition in a species is a prerequisite before using it as a biomarker to survey mercury in the environment. Abbreviations DLS, dynamic light scattering. FEBS Journal 274 (2007) 1849–1861 ª 2007 The Authors Journal compilation ª 2007 FEBS 1849 immune system, for which both autoimmunity and immune suppression have been reported [7–9]. Detec- tion of mercury in the environment is thus of high rele- vance for public health and in the framework of sustainable development. In this context, cholinesteras- es have been suggested as potential biomarkers and as the biological component of protein-based biosensors. The concept of biomarkers implies that in vivo choli- nesterase inhibition is measured after exposure of an animal to mercury, whereas in biosensors, inhibition takes place in vitro. For both applications, there is a need for high sensitivity of cholinesterases to mercury. Thus, it is a prerequisite to investigate the mechan- ism(s) of inhibition and to ascertain the reliability of using cholinesterases. Cholinesterases are believed to be sensitive to mer- cury; indeed, exposure of different organisms to suble- thal concentrations of mercury was shown to induce a significant decrease in cholinesterase activities in sev- eral organs [10–15]. However, uncertainties remain, as mercury-induced stimulation of cholinesterase activity has also been reported [16,17]. Several authors have described instantaneous reversible inhibition of choli- nesterases in vitro [18–20]. However, these findings could be artefactual, as activity measurements were often performed using Ellman’s reaction [21], the prod- ucts of which react with mercury and thus interfere with the measurement [22]. In addition, irreversible inhibition was described for Torpedo californica acetyl- cholinesterase, which leads in a first step to the forma- tion of a metastable state [23] that latter converts to a partially unfolded one [24]. Depending on their locations, two main functions have been ascribed to cholinesterases. At cholinergic synapses, cholinesterases are responsible for the ter- mination of nerve impulse transmission, by rapid hydrolysis of the neurotransmitter acetylcholine. This role is vital, as it allows restoration of neuronal excita- bility in cholinergic neuron networks. In noncholiner- gic tissues, cholinesterases belong to the group of ‘scavenger proteins’, which are responsible for the deg- radation of xenobiotics, e.g. succinylcholine or cocaine [25]. Playing these important roles, cholinesterases are among the most efficient enzymes in nature, with a substrate turnover of 10 3 )10 4 s )1 , depending on spe- cies [26]. Two types of cholinesterase are found in mammals, acetylcholinesterase and butyrylcholinest- erase, which are enzymatically distinguished by their substrate specificity. From the structural point of view, these enzymes are very similar, and only a few critical differences in the active site amino acid composition account for their differential behavior towards sub- strates [27–31]. Cholinesterases are % 60 kDa globular proteins, and are found in various oligomeric states. With regard to their cysteine content, three intrachain disulfide bonds are conserved, as well as another cysteine involved in intersubunit association. Although no free cysteine is found in some species, e.g. Electrophorus electricus acetylcholinesterase, there is one accessible to the bulk solution in most of them. Its position, however, is not conserved: 66 in human butyrylcholinesterase, 290 in Drosophila melanogaster acetylcholinesterase, and 231 in T. californica acetylcholinesterase. In the last case, the free cysteine has been shown to react with sulfhyd- ryl agents, resulting in irreversible inactivation of the enzyme [32,33]. In the present study, the kinetic mechanism of mer- cury-induced inactivation of cholinesterases was inves- tigated using four cholinesterases from various species to probe the potential variability in sensitivity that may exist in biomarkers used in ecotoxicologic studies. T. californica acetylcholinesterase, E. electricus acetyl- cholinesterase, D. melanogaster acetylcholinesterase and human butyrylcholinesterase were chosen because they are available in large amounts. Kinetic studies were complemented by X-ray crystallographic experi- ments on human butyrylcholinesterase and dynamic light scattering (DLS) studies on D. melanogaster acetylcholinesterase. Two inhibition mechanisms are proposed, depending on the presence or absence of a sensitive free cysteine. Results Inhibition of T. californica acetylcholinesterase Figure 1 shows the kinetics of irreversible inactivation of T. californica acetylcholinesterase by 1–10 lm HgCl 2 . Inhibition follows a pseudo-first-order kinetics (Scheme 1A; k i ¼ 9200 ± 480 m )1 Æmin )1 ), suggesting that inactivation involves only one site. Probably, this site is the same that has been shown to react with other thiols and organomercurial compounds [24,33], i.e. Cys231. Inhibition of human butyrylcholinesterase Incubation of 15 nm enzyme with HgCl 2 (1–10 mm HgCl 2 ) leads to rapid inhibition until a plateau is reached (Fig. 2A). Increasing the enzyme concentration diminishes the maximum inhibition (Fig. 2B). The inhibition appears to be slowly reversible; indeed, the 10-fold dilution of a sample incubated with 10 mm HgCl 2 leads to a slow increase of activity until a plat- eau is reached corresponding to the activity observed Cholinesterase inhibition by mercury M. F. Frasco et al. 1850 FEBS Journal 274 (2007) 1849–1861 ª 2007 The Authors Journal compilation ª 2007 FEBS after incubating the enzyme with 1 mm HgCl 2 (see sup- plementary Fig. S1A). To investigate this slow reacti- vation of the enzyme, human butyrylcholinesterase at a concentration of 15 nm was incubated with 1 or 10 mm HgCl 2 for 30 min. Samples were then dialyzed for 5 h (with a dilution factor of 1000), and activity was recorded as a function of time. A time-dependent reactivation of the enzyme was observed, suggesting that inhibition is reversible (Fig. 2C). Equilibrium is reached after 15–20 min (Fig. 2A), so the analysis of equilibrium between human butyrylcholinesterase and mercury was performed by incubating 15 nm enzyme for 30 min at different HgCl 2 concentrations. Subse- quently, the substrate o-nitrophenyl acetate was added, and the activity was measured. Data are best fitted by a model accounting for noncompetitive inhibition, with an apparent K iapp ¼ 0.4 ± 0.06 mm (Fig. 2D). Inhibi- tion of human butyrylcholinesterase by mercury thus occurs at a concentration 1000 times higher than that inhibiting T. californica acetylcholinesterase. Addition- ally, human butyrylcholinesterase inhibition by HgCl 2 can be described as an apparent, slow, noncompetitive reversible inhibition, which depends on the enzyme concentration, whereas the data for T. californica acetylcholinesterase can only be described as irrever- sible inhibition. Inhibition of D. melanogaster acetylcholinest- erase and E. electricus acetylcholinesterase D. melanogaster acetylcholinesterase and E. electricus acetylcholinesterase are inhibited by mercury in the same range of concentrations as human butyrylcholi- nesterase (i.e. 1000-fold higher than the concentration necessary to inhibit the Torpedo enzyme). In contrast to what was observed for human butyrylcholinesterase, inhibition of these two enzymes by HgCl 2 did not lead to a plateau, but rather showed a double exponential decay, as shown in Fig. 3A for D. melanogaster acetyl- cholinesterase (see supplementary Fig. S2 for E. electri- cus acetylcholinesterase). In addition, reactivation upon dilution is partial, restoring less than 10% of the initial activity (see supplementary Fig. S1B,C). As for human butyrylcholinesterase, however, inhibition decreases with enzyme concentration (Fig. 3B). Hence, inhibition of D. melanogaster acetylcholinesterase and E. electricus acetylcholinesterase by mercury can be described as a slow, noncompetitive, reversible process that depends on the enzyme concentration. However, an irreversible inhibition also takes place, which was not evident for human butyrylcholinesterase within 1 h of incubation with mercury. The D. melanogaster acetylcholinesterase used herein is recombinant; thus, it was possible to introduce sequence modifications by site-directed mutagenesis. To check whether this inactivation pattern was due to the free cysteine (C290) present on the surface of D. melanogaster acetylcholinesterase, this residue was mutated into an alanine. The resulting inhibition pat- tern was virtually identical to that of the wild-type enzyme, suggesting that this cysteine residue is not involved in the inhibition mechanism. Analogously, the alanine residue (A269) equivalent to the free cys- teine in position 231 of T. californica acetylcholinest- erase was mutated into a cysteine. In the micromolar range of HgCl 2 , this replacement also did not change the inhibition pattern of D. melanogaster acetylcholi- nesterase, strongly suggesting the involvement of the residues surrounding C231 in T. californica acetylcholi- B A ki E EM M k 3 E EM EM 2 K 0 k2 k1 M M D M M E 2 M 2 E E k6 k7 EM EM k5 k4 k 8 D Scheme 1. (A) Scheme proposed to describe the inhibition of T. californica acetylcholinesterase. E and M represent enzyme and mercury molecules, respectively, and the form EM is inactive. (B) Scheme proposed to describe the inhibition of D. melanogaster acetylcholinesterase. All forms are as active as the native enzyme, except for EM 2 and D, which are inactive upon mercury removal. Fig. 1. Remaining activity of T. californica acetylcholinesterase (7 n M) following incubation with mercury (1–10 lM). Curves corres- pond to a single multi-nonlinear fit of data for all concentrations of mercury in the equation derived from Scheme 1A [55]. M. F. Frasco et al. Cholinesterase inhibition by mercury FEBS Journal 274 (2007) 1849–1861 ª 2007 The Authors Journal compilation ª 2007 FEBS 1851 nesterase in its high sensitivity to mercurial agents. To check whether the introduction of a free cysteine inside the active site of D. melanogaster acetylcholinesterase would change the inhibition pattern, mutations F330C and Y370C were analyzed. These replacements did not change the inhibition pattern. Fig. 3. Remaining activity of D. melanogaster acetylcholinesterase following incubation with mercury. (A) Effect of mercury concentration with 500 n M enzyme. (B) Effect of protein concentration with 5 mM mercury. Fig. 2. Remaining activity of human butyrylcholinesterase following incubation with mercury. (A) Effect of mercury concentration with 15 nM enzyme. (B) Effect of protein concentration with 2.5 mM mercury. (C) Slow reactivation of inhibited enzyme following dialysis after 30 min of incubation with mercury. (D) Steady-state analysis of inhibition: enzyme and mercury were incubated for 30 min, after which the substrate o-nitrophenyl acetate was added to the cuvette at different concentrations, without significant dilution of the sample. Cholinesterase inhibition by mercury M. F. Frasco et al. 1852 FEBS Journal 274 (2007) 1849–1861 ª 2007 The Authors Journal compilation ª 2007 FEBS Structure of the HgCl 2 –human butyrylcholinesterase complex In this complex, four mercury-binding sites were char- acterized (Fig. 4A). These were attributed on the basis of very clear anomalous signals of mercury ions (Fig. 4B–E) at the employed wavelength (i.e. 1.54 A ˚ ). An isomorphous difference map, computed using the structure factors of the complex and those obtained from a native crystal (data not shown), confirmed these positions, displaying four very strong positive peaks overlapping with the anomalous peaks. In addi- tion, a pair of positive and negative densities was found in the active site, next to the catalytic serine residue (Ser198), on the atypical bond with the bound butyrate [31]. This feature was interpreted as a dis- placement of the butyrate upon complexation with mercury. No mercury ion was found in the active site. The first mercury-binding site (occupancy: 75%) was localized behind the ammonium-binding loci of the active site (Fig. 4B). At this site, mercury mainly inter- acts with His77Ne2 and Met81Sd (distances: 2.75 and 3.6 A ˚ , respectively), as well as with surrounding water molecules (distances: 2.96, 3.09, and 3.81 A ˚ , respect- ively). The second mercury ion (occupancy: 50%) binds to His423Nd1, Asn504Od1, and Thr505Oc1 (dis- tances: 2.33, 2.95, and 3.09 A ˚ , respectively; Fig. 4C). The third mercury ion (occupancy: 50%) is in close proximity to, and undergoes electrostatic interaction with, a sulfate anion (Fig. 4D) from the mother liquor solution (distance to the closest oxygen atom: 2.3 A ˚ ). The sulfate ion also interacts with His372Nd1 (distance to the closest oxygen atom: 2.5 A ˚ ), as has already been reported for the native structure. At these three previ- ously described loci, mercury binding occurs on the surface of the enzyme but does not involve crystal con- tacts. At the last binding site (occupancy: 25%), how- ever, a mercury ion was found at a special position in the crystal, in close proximity to the two Met511Sd (distance: 2.6 A ˚ ) residues of two symmetry-related molecules in the crystal (Fig. 4E). Hence, it is involved in crystal packing interactions. The structure did not show any mercury ion bound to a sulfhydryl group, as was observed in the case of T. californica acetylcholinesterase. However, the only free and accessible cysteine residue, Cys66, was per- sulfured (Cys-S-SH) in this batch of enzyme. Soaking of human butyrylcholinesterase crystals with mother liquor containing EDTA, dithiothreitol or l-cysteine did not allow reduction of the per-sulfur. Therefore, the potential binding of mercury to this cysteine ‘in solution’ remains an open issue. DLS assays At all mercury concentrations, data were fitted as only one species with a low polydispersity (polydispersity index ¼ 0.3). An increase in the hydrodynamic radius of D. melanogaster acetylcholinesterase was observed with increasing HgCl 2 concentrations (Fig. 5). Under the experimental conditions used (5 lm enzyme), the hydrodynamic radius increased linearly with mercury concentration. Physical changes and protein aggrega- tion occurred in the first minute after mercury addi- tion, and the size of the aggregate remained stable for at least 1 h. Kinetic model for D. melanogaster acetylcholinesterase inhibition by mercury D. melanogaster acetylcholinesterase was incubated with HgCl 2 for various times, and remaining activities were measured for 10 s following dilution (10-fold or 100-fold) of the sample. The incubation time varied from 30 s to 1 h, enzyme concentrations were 50, 100, 300, 500, 700 and 900 nm, and HgCl 2 concentrations were 1, 2.5, 5 and 10 mm. The 22 experimental curves were simultaneously analyzed with concurrent models, taking into account the information obtained from other experiments: (a) inhibition appears to be noncom- petitive, binding sites of mercury are located on the pro- tein surface, and inhibition does not involve residues in the active site; and (b) mercury binding promotes aggre- gation, and hence indirectly diminishes the enzyme sen- sitivity to mercury, most likely because aggregation reduces accessibility to the second mercury-binding site. Among all tested possibilities, Scheme 1B appears as the most simple and appropriate model to describe the irreversible and slow reversible inhibition of D. melano- gaster acetylcholinesterase by mercury (see kinetic constants in Table 1 and curve fitting in supplementary Fig. S3). According to this model, one mercury ion binds to the enzyme to form the complex EM (E and M represent enzyme and mercury molecules, respect- ively) with an equilibrium constant around 0.2 mm; this binding is instantaneously reversible and does not affect enzyme activity. The binding of a second mer- cury ion to the same enzyme molecule (EM) leads to an inactive form (EM 2 ). This inactivation is slowly reversible. In addition, this form is not stable and may result in irreversible enzyme denaturation (D). This part of the scheme describes the two phases of inhibi- tion. To describe the effect of protection by enzyme concentration, we introduced into the model the form E 2 M 2 , resulting from reversible aggregation of the form EM without any alteration of the enzymatic M. F. Frasco et al. Cholinesterase inhibition by mercury FEBS Journal 274 (2007) 1849–1861 ª 2007 The Authors Journal compilation ª 2007 FEBS 1853 BC ED Active-site gorge entrance A 1 2 3 4 Cholinesterase inhibition by mercury M. F. Frasco et al. 1854 FEBS Journal 274 (2007) 1849–1861 ª 2007 The Authors Journal compilation ª 2007 FEBS activity. This aggregate form (E 2 M 2 ) may either dena- turate (form D) or dissociate, thereby giving the reversible inactivated form (EM 2 ). Discussion Mercury inhibits cholinesterases The four cholinesterases analyzed in this study are inhibited by mercury, but through different mecha- nisms. Mercury in micromolar concentrations inhibits T. californica acetylcholinesterase, but the other tested cholinesterases are sensitive only in the millimolar range. Millimolar concentrations of mercury are irre- levant both under physiologic conditions and in the environment; indeed, concentrations up to 300 mgÆL )1 (% 1mm) have never been reported, even after bio- accumulation in highly polluted areas. The initial objective of this study was to evaluate the inhibition of cholinesterases by mercury, with a view to using them as biomarkers to survey polluted areas or for incorporation in biosensors. With regard to the utilization of cholinesterases as biomarkers, our work obviously demonstrates that the type and effect- iveness of inhibition of a cholinesterase by mercury strongly depend on the species. Therefore, the kinetic characterization of cholinesterase inhibition in the selected species would be a prerequisite for field stud- ies. A biosensor is an alternative to a biomarker, in that the enzyme is linked to a surface, deep in the sur- veyed solution, and inhibition of cholinesterase is recorded. Inhibition occurs in vitro, whereas it occurs in vivo in biomarkers. Cholinesterases as biological components were first developed to detect low levels of insecticides in the environment [34]. Numerous subse- quent studies have been performed to develop trans- ducers and to increase enzyme sensitivity and stability [35]; the biosensor technology for cholinesterases is therefore available, and permits consideration of their use for surveying mercury in the environment. Of the studied enzymes, it appears that only T. californica acetylcholinesterase could be considered a good candi- date, as it is the only one that is sensitive enough. Cholinesterase inhibition is not related to the active site Mercury inhibits a large number of enzymes with func- tional sulfhydryl group(s) in the active site [36–38]. This does not apply to cholinesterases, as: (a) there is no free cysteine in the active site; (b) the introduction of a free cysteine inside the active site of recombinant D. mel- anogaster acetylcholinesterase (F330C and Y370C) did not increase the sensitivity to HgCl 2 ; and (c) the com- plex structures of T. californica acetylcholinesterase Fig. 5. Aggregation of D. melanogaster acetylcholinesterase (5 lM) as a function of mercury concentration revealed by an increase in the hydrodynamic radius estimated by DLS. Fig. 4. Binding of mercury ions in the HgCl 2 –human butyrylcholinesterase complex. (A) Overview of mercury-binding sites on the surface of the enzyme. (B) First mercury-binding site (numbered ‘1’), next to His77 and Met81 (i.e. on the W-loop, behind the ammonium-binding loci of the active site ) Trp82). (C) Second mercury-binding site (numbered ‘2’), next to His423, Asn504 and Thr505. (D) Third mercury-binding site (numbered ‘3¢), next to a sulfate ion bound to His372. (E) Fourth mercury-binding site (numbered ‘4’), at a special position in the crystal, in proximity to Met511 of two symmetry-related molecules. The omit 2F o ) F c electron density map (contour level 1.5r), as well as the anomalous map (contour level 4r), are superimposed on the model in (B), (C), (D), and (E). Table 1. Kinetic constants describing D. melanogaster acetylcholi- nesterase inhibition by mercury according to Scheme 1B. Binding of the first mercury ion is treated as an instantaneous step (affinity is 0.2 m M). The fit was done simultaneously to all inactivation data (supplementary Fig. S3) and to the curve of reactivation data (sup- plementary Fig. S1B). To emphasize the reactivation data, they were weighted to the same y as the inhibition curves. K 0 (M) 2.07 ± 0.52 · 10 )4 k 1 (M )1 Æs )1 ) 681 ± 60 k 2 (s )1 ) 0.121 ± 0.015 k 3 (s )1 ) 0.00564 ± 0.00018 k 4 (M )1 Æs )1 ) 1.6 ± 0.3 · 10 7 k 5 (s )1 ) 0.0121 ± 0.017 k 6 (M )1 Æs )1 ) 1.6 · 10 7a k 7 (s )1 ) 0.0121 a k 8 (s )1 ) 0.0298 ± 0.0039 a k 6 was set identical to k 4 , and k 7 to k 5 ,ask 7 ¼ k 2 · k 5 ⁄ (k 2 + k 5 ) and k 2 ) k 5 . M. F. Frasco et al. Cholinesterase inhibition by mercury FEBS Journal 274 (2007) 1849–1861 ª 2007 The Authors Journal compilation ª 2007 FEBS 1855 ([24] ) Protein Data Bank accession code 2J4F) and human butyrylcholinesterase (this work ) Protein Data Bank accession code 2J4C) soaked in HgCl 2 failed to show mercury binding in their active site, at least with a dissociation constant lower than 10 mm. This obser- vation is surprising, considering the strong electrostatic dipole aligned with the gorge axis, which should attract positive charges within the active site [39]. The absence of mercury within the active site may be a consequence of its large solvation shell (I. Silman, unpublished results), which could prevent it from entering the gorge. The quaternary nitrogen of the substrate, on the other hand, is not hydrated [40], and can thus readily enter the gorge. The model we propose here for cholinest- erase inhibition by mercury is a rather general model for the effect of mercury on proteins; only in cases where a sensitive free sulfhydryl group is available should another model be considered, as, for example, in T. californica acetylcholinesterase. Mercury-binding sites on cholinesterases Organic and inorganic mercurials are capable of forming very tight bonds with functional groups such as thiolates of cysteine [41,42]. Sulfhydryl groups are considered to be the main targets of mercury, as they are the most reactive nucleophilic sites of protein amino acid side chains. HgCl 2 binds to a single resi- due to form R-S-Hg-Cl. Results for cholinesterase suggest that inactivation by mercury involves the free thiol group of Cys231. This functional group was shown to be the target of other sulfhydryl reagents, including organomercurials. Modifications of this resi- due lead to an irreversible inhibition of the enzyme that follows pseudo-first-order kinetics [24,33]. How- ever, introducing a cysteine at the same position in D. melanogaster acetylcholinesterase did not result in increased sensitivity to mercury, suggesting an import- ant role of the surrounding environment in the Torpedo enzyme. Mercury may also react with S–S bonds, leading to their disruption (R-S-Cl + Cl-Hg-S-R) [43]. The R-S-Cl moiety may later be oxidized by another mer- cury ion to form the compound R-S-Hg-Cl. The abil- ity of HgCl 2 to cleave S–S bridges enables it to disturb the tertiary structure of proteins and hence to lower their stability. In cholinesterases, three intra- chain disulfide bonds are conserved. Both site-directed mutagenesis of the cysteines involved in disulfide bond formation and cholinesterase treatment with reducing agents inactivate the enzyme [44], showing that these disulfides are essential for the protein to function. However, neither the complex structure with mercury of human butyrylcholinesterase, nor that of T. californica acetylcholinesterase [24], showed evidence of binding at these positions. Most likely, S–S bridges are too deeply buried inside the protein and thus are not accessible to the highly hydrophilic mercury. Metals are also capable of forming very tight bonds with histidine and methionine side chains as, for exam- ple, in metalloenzymes. In cholinesterases, mercurials were found to be linked to these residues, as evidenced by the complex structures with mercury of human butyrylcholinesterase (Fig. 4; Protein Data Bank entry 2J4C) and T. californica acetylcholinesterase (Protein Data Bank entry 2J4F). Mercury induces protein aggregation Aggregation induced by metal ions has been observed for other protein systems, particularly for proteins involved in protein deposition diseases [45–48]. DLS experiments have shown that binding of mercury ions promotes the aggregation of D. melanogaster acetyl- cholinesterase, perhaps as a consequence of the cross- linking of two coordinating residues present at the surface of the protein. This aggregation depends on protein concentration, and does not affect the folding of the protein; it may therefore protect the enzyme from unfolding, due to the binding of two mercury ions on the same enzyme molecule. Putative mechanism of inhibition A goal of this study was to address the issue of a clearcut model for cholinesterase inhibition by mer- cury. It appears that two different mechanisms for cholinesterase inhibition by mercury may be consid- ered. The first one, illustrated by the Torpedo enzyme (Scheme 1A), results from the binding of a mercury ion to a sensitive free cysteine and leads to irreversible inactivation. Similar mechanisms have been described for several proteins, e.g. the Na + –K + )2Cl – cotrans- porter, cystic fibrosis transmembrane conductance reg- ulator or urease [49–51]. The three other cholinesterases studied herein illus- trate the second mechanism of mercury-induced inhibi- tion. It would probably also be operative for T. californica acetylcholinesterase if the sensitive Cys231, which causes irreversible inactivation, was absent (Scheme 1B). Binding of the first mercury ion is instantaneous, with an equilibrium constant around 0.2 mm, and does not affect enzyme activity. The bind- ing of a second mercury ion, however, induces slow, reversible inactivation of the enzyme (EM 2 ), which can Cholinesterase inhibition by mercury M. F. Frasco et al. 1856 FEBS Journal 274 (2007) 1849–1861 ª 2007 The Authors Journal compilation ª 2007 FEBS promote irreversible unfolding (D). It may be proposed that mercury cross-links residues within the same enzyme molecule, thereby inducing a conformational change, which can lead to partial unfolding followed by irreversible denaturation. Mercury may also cross-link residues belonging to different molecules, leading to enzyme aggregation. As the model is operative if the resulting complex (E 2 M 2 ) is fully active, one can assume that these intermolecular cross-links do not induce conformational changes. Because inhibition decreases as protein concentration increases (Fig. 3B), it may be argued that the intermolecular cross-links protect the enzyme from the inactivating intramolecular ones. As residues involved in the binding of mercury at the surface are not conserved in the cholinesterase family, constants estimated for D. melanogaster acetyl- cholinesterase should vary for the other cholinesterases; for example, denaturation rate constants (k 3 and k 8 ) are anticipated to be lower for human butyrylcholinesterase than for D. melanogaster acetylcholinesterase, as inhibi- tion reaches a plateau and total reactivation was found following dilution. Experimental procedures Enzymes D. melanogaster acetylcholinesterase was produced in the baculovirus system and purified as previously described [52]. Mutants C290A, A269C, F330C and Y370C were obtained by site-directed mutagenesis. Mutations outside the active site (C290A and A269C) did not affect the enzyme activity, whereas an effect was observed with muta- tions inside the active site (F330C and Y370C; see supple- mentary Fig. S4). T. californica acetylcholinesterase was generously provided by I. Silman (Department of Neuro- biology, Weizmann Institute of Science, Rehovot, Israel). The native tetrameric E. electricus acetylcholinesterase and human butyrylcholinesterase used in the kinetic studies were obtained from Sigma (St Louis, MO, USA). The recombinant monomeric human butyrylcholinesterase used in the crystallographic study was produced and purified as previously described [53], and was generously provided by F. Nachon (Centre de Recherche du Service de Sante ´ des Arme ´ es, La Tronche, France). Cholinesterase inhibition Cholinesterases were diluted in Tris buffer (25 mm, pH 7.0), and ions that could have interfered with the solu- bility of mercury were removed by exclusion chromatogra- phy on a Sephadex G25 column (PD10; Amersham, Saclay, France). In order to ensure cholinesterase stability, BSA was added to a final concentration of 0.1 mgÆmL )1 , a con- dition in which no loss of activity was observed after sev- eral hours at 25 °C. Preliminary experiments showed that the impact of albumin on the toxic potency of mercury was negligible in the experimental conditions described herein, in accordance with previous reports [54]. For the analysis of inhibition time-courses, enzymes were incubated with inorganic mercury (HgCl 2 ) for different time periods, and residual activities were measured for 10 s, fol- lowing 10-fold or 100-fold dilutions. A stock solution of the substrate o-nitrophenyl acetate (1 m) was prepared in dimethylsulfoxide, and then diluted to a final concentration of 1 mm in the reaction buffer. The release of the enzymatic product o-nitrophenol was monitored spectrophotometrically by following its absorbance at 405 nm. At the concentra- tions reported in this study, no significant interference of mercury occurred with either the substrate o-nitrophenyl acetate or the product o-nitrophenol [22]. Analysis of equilibrium between human butyrylcholinest- erase and mercury was performed by incubating the enzyme with HgCl 2 for 30 min, after which the substrate o-nitro- phenyl acetate was added to the cuvette at different concen- trations without significant dilution of the sample. Human butyrylcholinesterase reactivation experiments were per- formed by dialysis using Slide-A-Lyzer Dialysis Cassettes of 10 000 molecular weight cutoff, 0.5–3 mL capacity (Pierce, Rockford, IL, USA). Experimental data were analyzed by multiple nonlinear regressions, using the fit program gosa (http://www. bio-log.biz). Data for Scheme 1A were analyzed using the solved explicit equations [55]. As integration of the differen- tial equations was too complex for equations corresponding to Scheme 1B, numerically solved systems of differential equations were fitted to the data [56]. Crystallization of human butyrylcholinesterase, soaking procedure, and data collection for the HgCl 2 –human butyrylcholinesterase complex Tetragonal crystals (space group I422) of recombinant monomeric human butyrylcholinesterase were obtained at 20 °C, using the hanging-drop vapor diffusion method. The mother liquor solution was composed of 2.1 m ammonium sulfate and 0.1 m Mes buffer (pH 6.5), and the protein con- centration was 6 mgÆmL )1 . As mercury induces protein aggregation and denaturation, we chose a crystal soaking procedure rather than cocrystallization to identify mercury- binding sites. A native human butyrylcholinesterase crystal was soaked for 30 min at 20 °C, in a mother liquor solu- tion containing 10 mm HgCl 2 . Prior to the flash cooling, the crystal was soaked for 20 s in a cryoprotective solution composed of 2.3 m ammonium sulfate, 0.1 m Mes buffer (pH 6.5), 10 mm HgCl 2 , and 18% glycerol. After flash-cool- ing of the crystal to 100 K in a nitrogen gas stream, X-ray diffraction data were collected on an in-house R-AXIS IV image plate detector installed on a Rigaku (Sevenoaks, M. F. Frasco et al. Cholinesterase inhibition by mercury FEBS Journal 274 (2007) 1849–1861 ª 2007 The Authors Journal compilation ª 2007 FEBS 1857 UK) rotating-anode generator. At the employed wavelength (k ¼ 1.54 A ˚ ), the anomalous signal of mercury ions permit- ted their unequivocal identification and localization. The dataset was indexed, merged and scaled using xds ⁄ xscale, and the amplitude factors were generated using xdsconv [57]. For further details, see Table 2. Structure determination and refinement The native structure of human butyrylcholinesterase (Pro- tein Data Bank entry code 1POI) without ions, water and sugar molecules was used as a starting model for rigid body refinement in the resolution range 20–4 A ˚ . Subsequently, the dataset underwent simulated annealing to 7500 K, with cooling steps of 10 K, followed by 250 steps of conjugate- gradient minimization. Diffraction data from 20 to 2.75 A ˚ were used for refinement, and maps were calculated using data between 15 and 2.75 A ˚ . All graphic operations, mode- ling and model building were performed with coot version 0.33 [58]. Minimization and individual B-factor refinement followed each stage of manual rebuilding. All refinements and map calculations were done using cns version 1.1 [59]. Structure refinements were evaluated using the procheck module [60] of the CCP4 suite [61]. Figure 4 was produced using pymol [62]. A summary of refinement statistics is shown in Table 2. DLS assays Dynamic light scattering (DLS) measurements were performed to assess aggregate formation in samples of D. melanogaster acetylcholinesterase incubated with HgCl 2 . Samples contained 5 lm enzyme prepared in Tris buffer (25 mm, pH 7.0) and various concentrations of HgCl 2 . Prior to measurements, enzyme and mercury solutions were filtered through 0.2 lm polyethersulfone membrane dispo- sable filters to ensure elimination of dust particles whose signal would interfere with that of protein molecules. Scat- tering data were collected for 60 min, at 20 °C, using a DynaPro MS ⁄ X instrument (Wyatt Technology, Santa Barbara, CA, USA). Recorded data were analyzed using dynamics autocorrelation analysis software (ver- sion 6, Protein Solutions, Wyatt Technology), which allowed us to obtain the median hydrodynamic radius and an estimate of the size distribution in the sample (polydis- perse index). Acknowledgements This work was partially supported by ‘Fundac¸ a ˜ o para a Cieˆ ncia e a Tecnologia’ and EU FEDER funds (M. F. Frasco PhD grant SFRH ⁄ BD ⁄ 6826 ⁄ 2001; pro- ject ‘CHOLINEOMANIA’ POCI ⁄ MAR ⁄ 58244 ⁄ 2004) and by bilateral cooperation projects Portugal ⁄ Slovenia (GRICES ⁄ Ministry of Education, Science and Sport, 2006) and Portugal ⁄ France (GRICES ⁄ EGIDE, Pessoa program, 2006). We are grateful to Professor Israel Silman and Dr Florian Nachon for the generous gifts of Torpedo californica acetylcholinesterase and recombinant monomeric human butyrylcholinesterase, respectively. Financial support by the CEA and the EMBO (ASTF230-2006) to M. Weik and J. P. Colle- tier, respectively, is gratefully acknowledged. We grate- fully acknowledge the ESRF for beamtime under long-term projects MX387 and MX498. Table 2. Data collection of the HgCl 2 –human butyrylcholinesterase complex. Human butyrylcholinesterase in complex with 10 m M HgCl 2 Protein Data Bank accession code 2J4C Temperature (K) 100 Oscillation step (°)1 Number of frames 120 Exposure time (min per frame) 20 Wavelength (A ˚ ) 1.54 Space group I422 Unit cell parameters (A ˚ ) a ¼ b 153.76 c 128.58 Resolution range (A ˚ ) 20.00–2.75 (2.80–2.75) a Completeness (%) 93.9 (97.8) R merge (%) b 6.9 (42.1) I ⁄ rI 20.97 (4.29) Unique reflections 35 972 Redundancy 4.36 Observations ⁄ parameters ratio 1.90 R cryst. (%) 16.26 R free (%) 21.95 rmsd bond length (A ˚ ) 0.0072 rmsd bond angles (°) 1.5027 rmsd with respect to native structure (A ˚ ) (Protein Data Bank accession code 1P0I) 0.1840 Number of atoms 4712 Protein 4176 Carbohydrate 118 Water 365 Ligands and ions 53 Wilson B factor (A ˚ 2 ) 42.6 Average B factor (A ˚ 2 ) 49.3 Protein 46.1 Carbohydrate 77.0 Water 63.5 Ligands and ions 78.3 a Values in paraentheses are for the highest resolution shell. b R merge ¼ R hkl R i jI i ðhklÞÀ<I ðHKLÞ >j R hkl R i I i ðhklÞ . Cholinesterase inhibition by mercury M. F. Frasco et al. 1858 FEBS Journal 274 (2007) 1849–1861 ª 2007 The Authors Journal compilation ª 2007 FEBS [...]... 10-fold or 100-fold dilution; o-nitrophenyl acetate at a final concentration of 1 mm was used as substrate (A) Human butyrylcholinesterase (B) Cholinesterase inhibition by mercury D melanogaster acetylcholinesterase (C) E electricus acetylcholinesterase Fig S2 Inhibition of E electricus acetylcholinesterase by mercury E electricus acetylcholinesterase (5 nm) was incubated with metal solutions for different... (1995) Inhibition of acetylcholinesterase activity in the central nervous system of the red swamp crayfish, Procambarus clarkia, by mercury, cadmium, and lead Bull Environ Contam Toxicol 55, 746–750 14 El-Demerdash FM & Elagamy EI (1999) Biological effects in Tilapia nilotica fish as indicators of pollution by cadmium and mercury Int J Environ Health Res 9, 173–186 Cholinesterase inhibition by mercury 15 El-Demerdash... 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RJ, Nwosu EC & Hanas JS (2001) Mercuric ion inhibition of eukaryotic transcription factor binding to DNA Biochem Pharmacol 61, 1543–1550 6 Mirzoian A & Luetje CW (2002) Modulation of neuronal nicotinic acetylcholine receptors by mercury J Pharmacol Exp Ther 302, 560–567 7 Lalancette A, Morin Y, Measures L & Fournier M (2003) Contrasting changes of sensitivity by lymphocytes and neutrophils Dev Comp Immunol... the inhibition of acetylcholinesterase activity in the freshwater fish Cyprinus carpio by mercury and zinc Biochem Int 26, 367–375 12 Schmidt GH & Ibrahim NM (1994) Heavy metal content (Hg2+, Cd2+, Pb2+) in various body parts: its impact on cholinesterase activity and binding glycoproteins in the grasshopper Aiolopus thalassinus adults Ecotox Environ Safe 29, 148–164 13 Devi M & Fingerman M (1995) Inhibition. .. inhibit acetylcholinesterase (AChE)? Implementation of assay conditions for the use of AChE activity as a biomarker of metal toxicity Biomarkers 10, 360–375 ˇ ´ 23 Sosˇ kic M, Pavlic MR & Zorko M (1979) Influence of ˘ some metals on acetylcholinesterase Iugosl Physiol Pharmacol Acta 15, 71–85 24 Kreimer DI, Dolginova EA, Raves M, Sussman JL, Silman I & Weiner L (1994) A metastable state of Torpedo californica... a final concentration of 1 mm as substrate Fig S3 Curve fitting with the proposed kinetic model for D melanogaster acetylcholinesterase inhibition by mercury The 22 experimental curves were simultaneously fitted using numerical integration, considering that data possessed a maximum of 10% error Enzyme concentrations were 50, 100, 300, 500, 700 and 900 nm, with four concentrations of HgCl2: (A) 1 mm; (B) . utilization of cholinesterases as biomarkers, our work obviously demonstrates that the type and effect- iveness of inhibition of a cholinesterase by mercury strongly. a clearcut model for cholinesterase inhibition by mer- cury. It appears that two different mechanisms for cholinesterase inhibition by mercury may be consid- ered.