Mechanisms of cholinesterase inhibition by inorganic mercury Manuela F. Frasco1,2, Jacques-Philippe Colletier3, Martin Weik3, Fe´ lix Carvalho4, Lu´ cia Guilhermino1,2, Jure Stojan5 and Didier Fournier6
1 ICBAS, Instituto de Cieˆ 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
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 HgCl2 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 (HgCl2) 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 HgCl2 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.
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
Abbreviations DLS, dynamic light scattering.
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Cholinesterases are (cid:2) 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].
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. it is a prerequisite to investigate the mechan- Thus, ism(s) of inhibition and to ascertain the reliability of using cholinesterases.
scattering (DLS)
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 studies on D. melanogaster light acetylcholinesterase. Two inhibition mechanisms are proposed, depending on the presence or absence of a sensitive free cysteine.
Results
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].
Inhibition of T. californica acetylcholinesterase
acetylcholinesterase by
Figure 1 shows the kinetics of irreversible inactivation 1–10 lm of T. californica HgCl2. Inhibition follows a pseudo-first-order kinetics (Scheme 1A; ki ¼ 9200 ± 480 m)1Æmin)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 HgCl2 (1–10 mm HgCl2) 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 HgCl2 leads to a slow increase of activity until a plat- eau is reached corresponding to the activity observed
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 103)104 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].
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an apparent Kiapp 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 HgCl2 can be described as an apparent, slow, noncompetitive reversible inhibition, which depends on the enzyme the data for T. californica concentration, whereas acetylcholinesterase can only be described as irrever- sible inhibition.
Inhibition of D. melanogaster acetylcholinest- erase and E. electricus acetylcholinesterase
Fig. 1. Remaining activity of T. californica acetylcholinesterase (7 nM) 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].
A
M
ki
E
E M
k8
D
E2M2
B
In addition,
E
k4
k6
k7
E M
EM
k5
E
M
M
k1
K0
k3
butyrylcholinesterase,
however,
EM
D
E
EM2
k2
M
M
(A) Scheme proposed to describe the inhibition of Scheme 1. 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 EM2 and D, which are inactive upon mercury removal.
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 HgCl2 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). reactivation upon dilution is partial, restoring less than 10% of the initial activity (see supplementary Fig. S1B,C). As for human 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.
thus,
after incubating the enzyme with 1 mm HgCl2 (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 HgCl2 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 HgCl2 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
The D. melanogaster acetylcholinesterase used herein it was possible to introduce is recombinant; 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 HgCl2, 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-
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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.
Fig. 3. Remaining activity of D. melanogaster acetylcholinesterase following incubation with mercury. (A) Effect of mercury concentration with 500 nM enzyme. (B) Effect of protein concentration with 5 mM mercury.
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.
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DLS assays
Structure of the HgCl2–human butyrylcholinesterase complex
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 HgCl2 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
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- the butyrate upon complexation with placement of mercury. No mercury ion was found in the active site.
residues of
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 in close proximity to the two Met511Sd the crystal, (distance: 2.6 A˚ ) two symmetry-related molecules in the crystal (Fig. 4E). Hence, it is involved in crystal packing interactions.
D. melanogaster acetylcholinesterase was incubated with HgCl2 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 HgCl2 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 (EM2). 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 E2M2, resulting from reversible aggregation of the the enzymatic form EM without any alteration of
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.
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1
2
Active-site gorge entrance
4
3
A
C
B
E
D
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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 ((cid:2) 1 mm) have never been reported, even after bio- accumulation in highly polluted areas.
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.
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 mM). 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.
)1Æs)1)
)1Æs)1)
2.07 ± 0.52 · 10)4 681 ± 60 0.121 ± 0.015 0.00564 ± 0.00018 1.6 ± 0.3 · 107
0.0121 ± 0.017
)1Æs)1)
1.6 · 107a 0.0121a 0.0298 ± 0.0039
K0 (M) k1 (M k2 (s)1) k3 (s)1) k4 (M k5 (s)1) k6 (M k7 (s)1) k8 (s)1)
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 it appears that only T. californica studied enzymes, acetylcholinesterase could be considered a good candi- date, as it is the only one that is sensitive enough.
a k6 was set identical to k4, and k7 to k5, as k7 ¼ k2 · k5 ⁄ (k2 + k5) and k2 (cid:3) k5.
Cholinesterase inhibition is not related to the active site
activity. This aggregate form (E2M2) may either dena- turate (form D) or dissociate, thereby giving the reversible inactivated form (EM2).
Discussion
Mercury inhibits cholinesterases
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 HgCl2; and (c) the com- plex structures of T. californica acetylcholinesterase
The four cholinesterases analyzed in this study are inhibited by mercury, but through different mecha-
Fig. 4. Binding of mercury ions in the HgCl2–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 2Fo ) Fc 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).
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acetylcholinesterase
[24],
that mercury of human butyrylcholinesterase, nor of T. californica 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.
its large solvation shell
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
([24] ) Protein Data Bank accession code 2J4F) and human butyrylcholinesterase (this work ) Protein Data Bank accession code 2J4C) soaked in HgCl2 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 (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
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
the surrounding environment
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. HgCl2 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 in the role of Torpedo enzyme.
Mercury may also react with S–S bonds,
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].
acetylcholinesterase
the
if
the cysteines
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 HgCl2 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 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
The three other cholinesterases studied herein illus- trate the second mechanism of mercury-induced inhibi- tion. for It would probably also be operative T. californica 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 (EM2), which can
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dition in which no loss of activity was observed after sev- eral hours at 25 (cid:2)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].
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 (E2M2) 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 (k3 and k8) 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.
For the analysis of inhibition time-courses, enzymes were incubated with inorganic mercury (HgCl2) 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].
Experimental procedures
Enzymes
Analysis of equilibrium between human butyrylcholinest- erase and mercury was performed by incubating the enzyme with HgCl2 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).
Crystallization of human butyrylcholinesterase, soaking procedure, and data collection for the HgCl2–human butyrylcholinesterase complex
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].
Cholinesterase inhibition
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).
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Tetragonal crystals (space group I422) of recombinant monomeric human butyrylcholinesterase were obtained at 20 (cid:2)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 (cid:2)C, in a mother liquor solu- tion containing 10 mm HgCl2. 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 HgCl2, 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, (25 mm, Cholinesterases were diluted in Tris buffer 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-
M. F. Frasco et al.
Cholinesterase inhibition by mercury
Table 2. Data collection of the HgCl2–human butyrylcholinesterase complex.
Human butyrylcholinesterase in complex with 10 mM HgCl2
2J4C 100 1 120 20
1.54
I422
Protein Data Bank accession code Temperature (K) Oscillation step ((cid:2)) Number of frames Exposure time (min per frame) Wavelength (A˚ ) Space group Unit cell parameters (A˚ )
a ¼ b c
DLS assays
153.76 128.58 20.00–2.75 (2.80–2.75)a 93.9 (97.8) 6.9 (42.1) 20.97 (4.29)
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.
35 972
4.36 1.90 16.26 21.95
0.0072 1.5027 0.1840
light scattering
Dynamic (DLS) measurements were performed to assess aggregate formation in samples of D. melanogaster acetylcholinesterase incubated with HgCl2. Samples contained 5 lm enzyme prepared in Tris buffer (25 mm, pH 7.0) and various concentrations of HgCl2. 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 (cid:2)C, using a (Wyatt Technology, Santa DynaPro MS ⁄ X instrument 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
Resolution range (A˚ ) Completeness (%) Rmerge (%)b I ⁄ rI Unique reflections Redundancy Observations ⁄ parameters ratio Rcryst. (%) Rfree (%) rmsd bond length (A˚ ) rmsd bond angles ((cid:2)) rmsd with respect to native structure (A˚ ) (Protein Data Bank accession code 1P0I) Number of atoms Protein Carbohydrate Water Ligands and ions Wilson B factor (A˚ 2) Average B factor (A˚ 2) Protein Carbohydrate Water Ligands and ions
4712 4176 118 365 53 42.6 49.3 46.1 77.0 63.5 78.3
.
a Values in paraentheses are for the highest resolution shell. b Rmerge ¼
Rhkl RijIi ðhklÞ(cid:4)
Structure determination and refinement
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.
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- ‘CHOLINEOMANIA’ POCI ⁄ MAR ⁄ 58244 ⁄ 2004) ject 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.
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The native structure of human butyrylcholinesterase (Pro- tein Data Bank entry code 1POI) without ions, water and
M. F. Frasco et al.
Cholinesterase inhibition by mercury
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Supplementary material
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 times, and residual activities were measured for 10 s, follow- ing 10-fold dilution using o-nitrophenyl acetate at 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 simulta- neously 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) 2.5 mm; (C) 5 mm; (D) 10 mm. In addition, the reactivation curve (supplementary Fig. S1B) was included in the fitting with an appropriate weighting. Fig. S4. Activity of Drosophila mutants: pS curves of wild-type and mutants of amino acids in the active site gorge.
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The following supplementary material online: Fig. S1. Enzyme reactivation following dilution. Enzy- mes were incubated with metal solutions for different times, and residual activities were measured for 10 s, following 10-fold or 100-fold dilution; o-nitrophenyl acetate at a final concentration of 1 mm was used (B) as substrate.
(A) Human butyrylcholinesterase.
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