Mutant of Bungarus fasciatus acetylcholinesterase with low affinity and low hydrolase activity toward organophosphorus esters

Biochimica et Biophysica Acta 1764 (2006) 1470 – 1478 www.elsevier.com/locate/bbapap

Mutant of Bungarus fasciatus acetylcholinesterase with low affinity and low hydrolase activity toward organophosphorus esters Thomas Poyot a,⁎,1, Florian Nachon a,1, Marie-Thérèse Froment a, Mélanie Loiodice a, Stacy Wieseler b , Lawrence M. Schopfer b, Oksana Lockridge b, Patrick Masson a a

Département de Toxicologie, Centre de Recherches du Service de Santé des Armées, BP 87, 38702 La Tronche Cedex, France b University of Nebraska Medical Center, Eppley Institute, 986805 Nebraska Medical Center, Omaha, NE 68198-6805, USA Received 30 March 2006; received in revised form 27 July 2006; accepted 27 July 2006 Available online 4 August 2006

Abstract Enzymes hydrolysing highly toxic organophosphate esters (OPs) are promising alternatives to pharmacological countermeasures against OPs poisoning. Bungarus fasciatus acetylcholinesterase (BfAChE) was engineered to acquire organophosphate hydrolase (OPase) activity by reproducing the features of the human butyrylcholinesterase G117H mutant, the first mutant designed to hydrolyse OPs. The modification consisted of a triple mutation on the 122GFYS125 peptide segment, resulting in 122HFQT125. This substitution introduced a nucleophilic histidine above the oxyanion hole, and made space in that region. The mutant did not show inhibition by excess acetylthiocholine up to 80 mM. The kcat/Km ratio with acetylthiocholine was 4 orders of magnitude lower than that of wild-type AChE. Interestingly, due to low affinity, the G122H/Y124Q/S125T mutant was resistant to sub-millimolar concentrations of OPs. Moreover, it had hydrolysing activity with paraoxon, echothiophate, and diisopropyl phosphofluoridate (DFP). DFP was characterised as a slow-binding substrate. This mutant is the first mutant of AChE capable of hydrolysing organophosphates. However, the overall OPase efficiency was greatly decreased compared to G117H butyrylcholinesterase. © 2006 Elsevier B.V. All rights reserved. Keywords: Acetylcholinesterase; Organophosphate hydrolase activity; Organophosphorus inhibitors; Molecular modelling; Site-directed mutagenesis; Slow-binding inhibition

1. Introduction Organophosphorus anticholinesterases (OPs) are among the most toxic compounds synthesized by man. Originally, OPs were developed for use as insecticides, but their extreme toxicity toward vertebrates has led to malicious development of certain OPs as chemical warfare agents. Though the threat of the use of OPs in warfare is diminishing, the risk of chemical terrorist attacks has greatly increased in recent years. These Abbreviations: AChE, acetylcholinesterase; ATC, acetylthiocholine; BChE, butyrylcholinesterase; BTC, butyrylthiocholine; ChE, cholinesterase; DFP, diisopropyl phosphofluoridate; DTNB, dithio-bis-nitrobenzoic acid; OP, organophosphorus; OPase, organophosphorus hydrolase; TNB, 5-thio-2nitrobenzoic acid ⁎ Corresponding author. Tel.: +33 4 76 63 97 44; fax: +33 4 76 63 69 62. E-mail address: [email protected] (T. Poyot). 1 These authors contributed equally to this work. 1570-9639/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2006.07.008

toxic phosphoesters have a very high potency because they react rapidly with the active-site serine of cholinesterases (ChEs). Loss of acetylcholinesterase (AChE, EC. 3.1.1.7) physiological function leads to muscle paralysis, seizures and apnea followed by death [1]. Prophylaxis of OP poisoning is based on chemical protection of AChE by carbamylation. Current emergency therapy consists of counteracting the muscarinic effects of AChE inhibition, reactivating phosphylated AChE (i.e., phosphorylated or phosphonylated), and stopping seizures. Enzymes capable of degrading OPs are now emerging as a safe and efficient alternative to pharmacological approaches. Biological scavengers, either stoichiometric or catalytic, represent potential candidates for pre-treatment, treatment and decontamination of skin, mucosa and open wounds [2–4]. Given that slow spontaneous reactivation may occur for some OP-ChE conjugates, rational design of self-reactivating

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ChEs was proposed. An extensive work was made on human butyrylcholinesterase (hBChE, EC. 3.1.1.8) to design and express mutants with organophosphate hydrolase (OPase) activity [5–8]. hBChE was chosen first because it reacts with a broad range of OPs. Indeed, comparative substrate specificities [9,10], molecular models and later its 3D structure [11] showed that hBChE has a larger active site pocket than AChE. The mutants of hBChE were first designed with the aim of introducing a nucleophilic residue close to the adduct phosphorus atom. Mutating Gly117 of the oxyanion hole at position 117 into a histidine (G117H) or an aspartate (G117D) confers OP hydrolase activity to the enzyme [6,8]. Similarly, the natural mutation glycine to aspartate at the equivalent position was found to provide OP hydrolase activity to Lucilia cuprina carboxylesterase [12]. The OPase activity of G117H hBChE is the highest among these mutants, however it is too low for therapeutic use. The G117H mutant showed hydrolysing activity against paraoxon, sarin and VX, but not soman because of rapid dealkylation of the OP adduct (aging). Hydrolysis of soman was possible by introducing an additional mutation, E197Q, to slow down dealkylation of the adduct [7]. The molecular mechanism behind G117H hBChE OPase activity is not precisely determined. It has been suggested that histidine in position 117 acts as a hydrogen-bond acceptor to activate a water molecule, which subsequently hydrolyses the adduct [8]. However, molecular modelling suggests that the imidazole of His117 and the adduct phosphorus atom are in close contact, thus not allowing enough room for a water molecule. Therefore, transfer of the phosphyl moiety to His117 followed by hydrolysis of the phosphyl-histidine may be a more likely scenario. In the present work, we followed the same approach, introducing the nucleophile histidine at the same position in Bungarus fasciatus AChE in order to convert it into an organophosphate hydrolase with high efficiency. Singly substituted AChE, containing only the His mutation at position 122 (equivalent to position 117 in hBChE), had no

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activity with ATC. Therefore, multiple mutations were introduced. Bungarus fasciatus AChE (BfAChE) was chosen because it is easily expressed at high levels in mammalian cells as a monomer [13]. Analysis of the active site using molecular modelling tools suggested that if a histidine were introduced in place of Gly122, it would distort the active site due to steric hindrance. Thus, the replacement of Tyr124 by a smaller residue was expected to avoid active site distortion. This was finally achieved by mimicking G117H hBChE, i.e., substituting the complete peptide segment 122GFYS125 by 122HFQT125 in BfAChE. Residue F123 is present in both peptide segments, therefore we refer to this mutant as HQT. Energy minimized models of Bungarus fasciatus AChE G122H/Y124Q/S125T mutant (HQT) and of human BChE G117H mutant are shown in Fig. 1. The catalytic properties of HQT BfAChE mutant toward ATC and three OPs are described. Though the OPase activity is low, the engineered enzyme is the first AChE capable of hydrolysing OPs. 2. Experimental 2.1. Chemicals Acetylthiocholine (ATC) iodide, demeton, dithio-bis-nitrobenzoic acid (DTNB), paraoxon and buffer components of biochemical grade were purchased from Sigma (St. Louis, MO, USA). Diisopropyl phosphofluoridate (DFP) was purchased from Acros (Geel, Belgium), chlorpyrifos-oxon from CIL Cluzeau (Puteaux-La Défense, France) and echothiophate from Biobasal (Basel, Switzerland). 2.2. Construction and expression of the mutants The HQT mutants were made by PCR using Pfu polymerase and cloned into the mammalian expression plasmid pGS [5–8]. Stably transfected CHO-K1 cells (American Type Culture

Fig. 1. Active site of hBChE (A), G117H hBChE (B), Bungarus fasciatus AChE (C) and G122H/Y124Q/S125T (HQT) Bungarus fasciatus AChE (D). Oxygen atoms are in red, nitrogen atoms are in blue and carbon atoms are in green. Ser198 (A and B) and Ser203 (C and D) are the active site serines. Ala277 and Trp282 mark the entrance to the active site gorge. The area occupied by G122H/Y124Q/S125T mutation is highlighted in blue. Mutated residues are labeled in red. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Collection, N° CCL61) were selected in 50 μM methionine sulfoximine. The G117H mutant of human butyrylcholinesterase was expressed in CHO cells [5,6]. 2.3. Enzyme purification The enzymes were purified in 2 steps. Six liters of culture medium were passed over a 200-mL sepharose-4B procainamide affinity gel column (GE Healthcare, UK). The enzymes were eluted with 300 mL 0.3 M NaCl/0.3 M tetramethylammonium bromide in 50 mM phosphate pH 7.0. The eluate was dialysed against 25 mM Tris/HCl pH 8.0 buffer to remove the salts. In a second step, 50 ml of the dialysate was chromatographed on a Mono Q ion exchange column (GE Healthcare, UK) and eluted with 0.35 M NaCl, 25 mM Tris/HCl pH 8.0. The enzyme concentration, [E], was estimated for snake AChE from protein concentration as assayed with the bicinchoninic acid kit (Pierce) and comparison of activity and protein staining on non-denaturing gels [14]. The enzyme was estimated to be > 95% pure and homogenous and it was assumed to be fully active. The G117H mutant of human butyrylcholinesterase was purified on a procainamide affinity column, followed by ion exchange chromatography on a ProteinPak Waters Millipore HPLC column. A specific activity of 150 units/mg, as measured with 1 mM butyrylthiocholine, represented 100% pure enzyme [6]. 2.4. Kinetics of substrate hydrolysis

v¼

 
kcat ½E 1 þ b½S=Kss 1 þ Km =S 1 þ ½S=Kss

2.5. Comparison of the hydrolysis of ATC by wild type and HQT BfAChE in presence of 100 μM organophosphate ester Stock solution of echothiophate was 1 M in water. Stock solutions of paraoxon and DFP were respectively 1 M and 0.4 M in anhydrous MeOH. Stock solutions of demeton, and chlorpyrifos-oxon were 0.1 M in anhydrous MeOH. Hydrolysis of 1 mM ATC in 0.1 M phosphate buffer, pH 7.0 and 25 °C with 0.1% bovine serum albumin was followed at 420 nm using Ellman's method [15]. The initial rate for both wild type and HQT BfAChE was recorded for 2 min. Then 100 μM paraoxon, echothiophate, demeton, chlorpyrifos-oxon or DFP was added to the cuvette. The activity was recorded for an additional 10 min. 2.6. Hydrolysis of ATC by HQT BfAChE in presence of high concentration of paraoxon and echothiophate

Kinetic measurements were carried out using acetylthiocholine as substrate. All assays were carried out at pH 7.0, as a compromise between the optimal pH of enzyme activity and the stability of OPs in solution. All assays were made in triplicate. Hydrolysis of ATC was measured at 420 nm by the Ellman method [15] in 0.1 M sodium phosphate buffer pH 7.0 at 25 °C. Buffers were supplemented with 0.1% (w/v) bovine serum albumin as an enzyme stabilizer. The final concentration of Ellman's reagent, dithio-bis-nitrobenzoic acid (DTNB) in the cuvette was 0.35 mM. Wild-type snake AChE displays inhibition by excess substrate with ATC [13]. This phenomenon is described by the mechanistic model of Radic [10] and its following rate equation:


SIGMAPLOT 4.16 software (Jandel Scientific, San Rafael, CA, USA). When the data corresponded to Michaelis–Menten behaviour, Km and Vmax were determined by simple weighted nonlinear regression of the Michaelis–Menten equation, i.e., the first part of Eq. (1) using the SIGMAPLOT 4.16 software. For the HQT mutants, titration of active sites was not possible using a phosphorylating reagent. Therefore, enzyme concentration was estimated from protein concentration as described under enzyme purification, assuming that 100% of the catalytic sites of pure enzyme were active, kcat therefore represents the minimum theoretical kcat.

Hydrolysis of 1 mM ATC by HQT BfAChE with 1–8 mM paraoxon or 10–200 mM echothiophate in 0.1 M phosphate buffer, pH 7.0 and 25 °C, was followed at 420 nm using Ellman's method [15]. As the product of hydrolysis of paraoxon and echothiophate could interfere with the Ellman's test, we controlled that in absence of ATC, no activity was detectable. Spontaneous hydrolysis rates of ATC were subtracted from enzyme-catalyzed rates. In a first set of experiments, the activity was monitored upon simultaneous mixing of the enzyme, substrate and organophosphate. In a second set of experiments, the enzyme was incubated for 15 min with the organophosphate prior to adding ATC and recording the activity. 2.7. Hydrolysis of organophosphate esters by HQT BfAChE and G117H hBChE

ð1Þ

Km is the Michaelis constant, kcat the catalytic constant, [E] the enzyme active-site concentration and Kss the dissociation constant of the ternary SES complex. The parameter b reflects the efficiency by which the ternary complex SES forms product. When b > 1, there is substrate activation, when b < 1, there is substrate inhibition; for an enzyme that obeys Michaelis– Menten model, b = 1. Catalytic parameters Km, Kss and b values were calculated by nonlinear regression of Eq. (1): using the

Hydrolysis of paraoxon, echothiophate and DFP by mutated BfAChE and G117H hBChE was carried out at 25 °C. Spontaneous hydrolysis rates of OPs were subtracted from enzyme-catalyzed rates. Paraoxon hydrolysis was directly monitored in 0.1 M sodium phosphate buffer pH 7.0 at 400 nm, by measuring the release of its hydrolysis product, p-nitrophenol. Echothiophate iodide hydrolysis was followed in 0.1 M sodium phosphate buffer pH 7.0 in the presence of 0.35 mM DTNB, by recording the absorbance increase of 5-thio-2-benzoic acid (TNB) at 420 nm.

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DFP hydrolysis was measured using an ionometer equipped with a fluoride-specific electrode (Radiometer analytical SAS, 69100 Villeurbanne, France), to follow the release of the fluoride product. In this case, 0.1 M phosphate buffer (with 0.1% bovine serum albumin) at pH 6.0 was used, in order to operate at the optimal pH of the electrode. The concentration of stock solution of BfAChE was estimated using the bicinchoninic acid assay as described above. The concentration of G117H hBChE was calculated from its BTC activity and the kcat for BTC from the literature [6]. 3. Results 3.1. Catalytic properties of snake mutated AChE Table 1 shows the catalytic properties of wild-type BfAChE and HQT mutant, with acetylthiocholine as the substrate. Wildtype BfAChE showed excess substrate inhibition (b = 0.46; Kss = 36 mM) as previously described [13,16]. By contrast, BfAChE HQT mutant displayed apparent Michaelis–Menten behaviour; no excess substrate inhibition was observed with ATC up to 80 mM. Its catalytic properties were dramatically altered. The kcat/Km ratio for ATC (0.52 × 106 M− 1 min− 1) was decreased by 4 orders of magnitude compared to the wildtype enzyme. This drop is mainly due to the decrease of affinity (Km = 34 ± 4 mM instead of 0.052 ± 0.003 mM for the wild-type). Noteworthy, when the same set of mutations was introduced into human AChE, the enzyme had also a drop of Km (8.6 ± 1.6 mM vs. 0.1 mM for the wild-type [17]) and lost inhibition by excess substrate. As the human AChE HQT mutant did not present better catalytic properties than BfAChE HQT mutant, it was not further studied. 3.2. Effects of echothiophate, paraoxon, chlorpyrifos-oxon and demeton on HQT BfAChE We studied the effects of different classes of OPs on HQT BfAChE: insecticides (paraoxon, demeton, chlorpyrifos-oxon), glaucoma treatment (DFP, Echothiophate) (Fig. 2) Hydrolysis of 1 mM ATC by wild-type BfAChE was completely inhibited by 100 μM echothiophate, paraoxon, chlorpyrifos-oxon, DFP or demeton within seconds after mixing. On the other hand, the HQT BfAChE mutant was not inhibited under the same conditions for up to 10 min after the mixing, i.e., the mutant enzyme is resistant to OPs. It was important to further characterise the type of interaction of OPs with HQT BfAChE by increasing the concentration of OPs. We focused on 3 representative OPs: echothiophate, paraoxon and DFP.

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In presence of echothiophate at concentrations in the millimolar range (10–200 mM), the rate of hydrolysis of 1 mM ATC was identical with or without 15-min preincubation of the enzyme, meaning there was no progressive inhibition. However, echothiophate behaved like an apparent competitive inhibitor with a calculated Ki of 80 ± 14 mM. This result does not preclude the possibility that echothiophate is hydrolysed by HQT BfAChE mutant. Indeed, competitive inhibition is expected if echothiophate binds rapidly and reversibly to HQT BfAChE and if the hydrolysis rate of echothiophate is much slower than the hydrolysis rate of ATC. We also investigated the effects of paraoxon on the hydrolysis of 1 mM ATC further. We observed a surprising, slight and reproducible activation of ATC hydrolysis for paraoxon in the millimolar range (Fig. 3). At 8 mM paraoxon, the rate of hydrolysis increased by 20% compared to the control. We could not investigate the effect of higher concentrations of paraoxon, because of its limited solubility in aqueous solutions (< 10 mM). 3.3. Competition between DFP and ATC hydrolysis for BfAChE HQT mutant To investigate the mechanism of interaction between HQT mutant and DFP, a series of measurements was initiated by adding the enzyme to mixtures of 1 mM ATC and 2–20 mM DFP. The concentration of ATC was well below the Km for HQT BfAChE (34 mM). The release of TNB was monitored at 420 nm for 20 min. Fig. 4A shows progress curves for hydrolysis of 1 mM ATC in the presence of increasing concentrations of DFP. Simple inspection of Fig. 4A revealed that the rate of ATC hydrolysis decreased slowly over the first 6 min of the time course, ultimately reaching steady state. The steady state rate decreased as DFP concentrations increased. The existence of a burst-like induction time in progress curves (from 180 s at 2 mM DFP to 50 s at 20 mM DFP) suggested that inhibition of HQT mutant by DFP obeys a slow-binding mechanism. The induction rate constant k was determined by fitting the progress curves to the following equation: P ¼ vss d t þ

 vi  vss  d 1  ekd t k

ð2Þ

Where P is the concentration of released product (thiocholine) at time t, vss is the ultimate steady state rate at t → ∞, vi is the initial rate, and k is the apparent first order rate constant which describes the approach to steady state. We used GOSA, a simulated annealing based fitting software [18] (BioLog, Toulouse, France; http://www.bio-log.biz).

Table 1 Catalytic properties of wild-type and HQT mutant of Bungarus fasciatus AChE with acetylthiocholine as the substrate Wild-type ⁎ HQT mutant

Km (mM)

kcat (min− 1)

〈kcat/Km〉 (10−6 M− 1 .min− 1)

b

0.052 ± 0.003 34 ± 4

265 000 ± 39 700 17 600 ± 1500

5100 0.52

0.46 1

Kinetic measurements were carried out at 25 °C in 0.1 M phosphate buffer, pH 7.0. ⁎ kcat, Km and b values for WT BfAChE are from the literature [16].

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Fig. 2. Organophosphate compounds used in this study.

Three types of slow binding inhibition have been described which yield time courses of this type [19,20]. The time course for slow-binding inhibitors conforms to the general Eq. (2). The three mechanisms can be distinguished from the dependence of k on inhibitor concentration. k was obtained, for various concentrations of DFP, by nonlinear fitting of progress curves to Eq. (2). Fig. 4B shows an increasing hyperbolic dependence of k on [DFP]. This indicates that binding of the inhibitor to the enzyme active site obeys mechanism B of slow binding inhibition (Scheme 1) [19,20]. In this mechanism, there is an initial rapid formation of an EY complex, which then undergoes a slow isomerization to EY*. Competition becomes progressively more pronounced as EY is slowly converted to EY*. Assuming that [Y] and [S] >> E0, then vi, vss and k (from Eq. (2)) are given by: k dE d½S 2 0   ð3Þ vi ¼  Km d 1 þ K½YY þ ½S  k2 dE0 d½S    þ ½ S  Km 1 þ ½Y d KKYEYdKþ1 EY k2 dE0 d½S   ¼

½Y  Km 1 þ þ ½S  k4

vss ¼ 

0

KY þ k

noted that all concentrations of substrate, ATC, used in these experiments were very low relative to Km (where Km = 34 mM from Table 1). According to Eq. (4), when 1/vss = 0 then Y = −KY.KEY/(1 + KEY)*(1 + [S]/Km), so that KY.KEY/(1 + KEY)*(1 + [S]/Km) is given by the intercept on the x-axis in the plot 1/vss versus Y. At times greater than 6 min, the reaction of HQT BfAChE, ATC and DFP reached the final steady state rate, allowing vss to be measured. Simultaneous fitting of 1/vss versus [DFP] at 3 different ATC concentrations (1.5–3 mM) gave KY.KEY/(1 + KEY)* (1 + [S]/Km) = 0.44 ± 0.02 mM (Fig. 5). Since [S] << Km, KY.KEY/ (1 + KEY)*(1 + [S]/Km) reduces to KY.KEY/(1 + KEY). It is interesting to note that KY.KEY/(1 + KEY) is the apparent inhibition constant (Ki,app) for reversible inhibition of enzyme for HQT BfAChE by DFP. A second set of experiments was performed for the purpose of obtaining a value for KY.KEY/(1 + KEY). The steady-state velocity, vss, was measured after 6-min incubation of the enzyme with various mixtures of (1.5, 2.0 and 3.0 mM) ATC and DFP (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mM). The ATC

ð4Þ

4 þk4

1 ½Y    þ KEY A k ¼ k4 @ ½Y  þ KY 1 þ K½Sm k4 d½Y    þ k4 ¼ ½Y  þ KY 1 þ K½Sm

ð5Þ

With E0 as the total enzyme concentration, Km = (k− 1 + k2)/k1, KY = k− 3/k3 and KEY = k− 4/k4 (where KEY = [EY]/[EY*] at the ultimate steady state). Non-linear fitting of k versus [DFP] data from Fig 3A to Eq. (5) (where DFP = Y) did not yield accurate values for equilibrium constants KY and KEY. In order to improve the accuracy, it was necessary to use another relation linking KY and KEY as a constraint to the non-linear fitting. Such relation was derived from the plot 1/vss versus [Y]. It should be

Fig. 3. Stimulatory effect of paraoxon on ATC hydrolysis by HQT BfAChE. Progress curves for the hydrolysis of 1 mM ATC in the absence and in presence of 8 mM paraoxon were monitored in 0.1 M phosphate buffer, pH 7.0, at 25 °C. Enzyme was pre-incubated with paraoxon for 15 min, and then the reaction was started by adding ATC. This assay, as other assays, was performed in triplicate; data presented here correspond to a representative assay.

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Scheme 1.

Using KY.KEY/(1 + KEY) = 0.44 ± 0.02 mM as a constraint, k4, KY and KEY were determined by non-linear fitting from Eq. (5). The values are k4 = 1.80 ± 0.24 min− 1, KY = 10.9 ± 0.7 mM and KEY = 0.043 ± 0.013. 3.4. Hydrolysis of OPs by HQT Bungarus fasciatus AChE mutant

Fig. 4. Competition between DFP and ATC hydrolysis for BfAChE HQT mutant. (A) Progress curves for the hydrolysis of 1 mM ATC in the presence of various concentrations of DFP (4–20 mM), were monitored in 0.1 M phosphate buffer, pH 7.0, at 25 °C. For clarity of the figure, only a few points from the raw data are presented. (B) Apparent first order rate constant k as a function of DFP concentration. The curve represents the fit of data using Eq. (5).

concentrations are low relative to Km. Ki,app = KY.KEY/(1 + KEY) was determined by simulated annealing fitting of the plots 1/vss versus [DFP] to Eq. (4). k4, KY and KEY were determined by simulated annealing fitting of k versus [DFP] to Eq. (5) with the value of KY.KEY/(1 + KEY) as a constraint, using GOSA.

Echothiophate, paraoxon and DFP not only bind to HQT BfAChE but are slowly hydrolysed by the enzyme. However, the hydrolysis rate is so slow that the reversible binding of these compounds translates into apparent inhibition or activation. Large amounts of enzyme and long incubation times were required to study this slow hydrolysis. We compared the catalytic efficiency of HQT BfAChE to G117H hBChE for hydrolysis of echothiophate, paraoxon and DFP. The rate of hydrolysis for each organophosphate was measured by monitoring product release, see Experimental. Table 2 presents the relative efficiency of the 2 enzymes for echothiophate, paraoxon and DFP at 1 mM. In analysing the data, the organophosphates were treated as substrates, and it was assumed that [S] << Km for HQT BfAChE so that: v kcat ¼ d ½S  E Km

ð6Þ

In contrast, based on values from various OPs like sarin (110 μM), VX (50 μM), paraoxon (70 μM) and echothiophate (74 μM) [6], the Km of G117H hBChE for DFP was reasonably assumed to be about 100 μM. Hence, it was assumed that [S]>>Km for G117H hBChE so that: v ¼ kcat E

Fig. 5. Determination of KY.KEY/(1 + KEY) from the plot 1/vss versus DFP. The plotted lines represent the reciprocal value of the vss parameter obtained for 3 different ATC concentrations. KY = k− 3/k3 is the equilibrium constant between E and EY, and KEY = k− 4/k4 is the ratio between EY and EY* at the final steady state (See Scheme 1).

ð7Þ

values for v/E for HQT BfAChE, together with the [S] give kcat/ Km for HQT BfAChE. Values of v/E for G117H hBChE together with the Km values for G117H, give access to kcat/Km for G117H. The ratio (kcat/Km)G117H/(kcat/Km)HQT reflects the relative efficiency of HQT BfAChE compared to the G117H mutant of hBChE. The efficiency of OP hydrolysis by G117H was higher than by HQT: 420 times for echothiophate, 90 times for paraoxon, and about 7 times for DFP. Reported kcat values for G117H with paraoxon and echothiophate are 0.75 min− 1, and Km values are 0.07 mM for paraoxon and 0.074 mM for echo-

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Table 2 Comparison of kcat/Km ratios for paraoxon, echothiophate and DFP with HQT BfAChE and G117H hBChE mutants 〈kcat/Km〉 (min− 1 M− 1)

Paraoxon Echothiophate DFP

G117H hBChE

HQT BfAChE

Relative 〈kcat/Km〉 ðkcat =km ÞG117H ratio ðkcat =km ÞHQT

5700 10 100 5200

64 24 760

90 420 7

Kinetic measurements were carried out at 25 °C in 0.1 M phosphate buffer, pH 7.0 with paraoxon and echothiophate, and in 0.1 M phosphate buffer, pH 6.0 for DFP. Assays were performed in 3–5 times with standard deviations of 5–20%. For HQT BfAChE, it was assumed that [S] << Km so that kcat/Km ratio was obtained using Eq. (6). For G117H hBChE, it was assumed that [S] >> Km so that kcat/Km ratio was obtained using Eq. (7) and values taken from the literature.

thiophate [6], i.e., kcat/Km = 10 560 min− 1 M− 1 for paraoxon and 10 130 min− 1 M− 1 for echothiophate. We obtained a similar value for echothiophate (kcat/Km = 10 100 min− 1 M− 1). The difference between paraoxon values (kcat/Km = 5700 min− 1 M− 1 against 10 130 min− 1 M− 1) comes from the inaccuracy in our estimation of kcat (0.4 min− 1 instead of 0.75 min− 1).

4. Discussion 4.1. Catalytic properties with acetylthiocholine The HQT BfAChE mutant's catalytic efficiency with ATC as the substrate is severely decreased compared to wild-type AChE (4 orders of magnitude for the kcat/Km ratio). The magnitude of decrease is greater for its affinity (650-fold) than for its activity (15-fold). This was not the case for the G117H mutant of hBChE where the Km value for butyrylthiocholine (BTC) was only one order of magnitude lower compared to the wild-type [6]; kcat/Km for BTC decreased by 11-fold for the G117H, and decreased by 110-fold for the G117H/E197Q [7]. So, the reorganized active site of the HQT BfAChE is far less efficient than the one in G117H hBChE. The decrease of kcat may be due to a change in the lining of the aromatic residues in the active site gorge. The aromatic residues have been described to control the optimal position of the catalytic histidine for hAChE [21]. These residues are conserved in BfAChE and the triple HQT mutation, notably Y124Q, is expected to disturb this lining. HQT BfAChE mutant follows apparent Michaelis–Menten kinetics up to 80 mM ATC at pH 7.0. It is not inhibited by excess substrate. At physiological pH, excess cationic substrate usually causes inhibition of AChE, whereas it causes activation of BChE. Noteworthy, substrate inhibition is pH dependant [16]. The mechanistic model of Radic [10] and its rate Eq. (1) describe these particularities. At high substrate concentration a molecule of substrate binds to the peripheral site, slowing down the association and dissociation of substrate at the acylation site [22]. Rosenberry's group proposed that the changes in the binding of substrates at the acylation site is due to a steric blockade by substrate bound at the peripheral site. The crystal structure of Torpedo californica in presence of high concentra-

tion of acetylthiocholine recently confirmed this hypothesis [23]. No steric blockade can be achieved when substrate fully equilibrates with the active site. Given that the conformational integrity of the active site of BfAChE is affected by the triple HQT mutation resulting in low kcat/Km for ATC, substrate would be expected to equilibrate and this is the reason that substrate inhibition is lost. In contrast hBChE G117H displays the same level of activation by excess substrate (b = 3) as the wild-type enzyme [6]. This shows that the introduction of a histidine in hBChE's active site, does not significantly disturb association and dissociation of substrate. 4.2. Interaction with OPs As a consequence of the likely disorganization of its active site architecture, HQT BfAChE binds the OPs we tested poorly and is therefore strongly resistant to inhibition by OPs. Interestingly, insect AChEs with natural mutations at the homologous glycine were reported to acquire resistance to carbamates. For example, G247S mutation in AChE (p-Ace) from Culex pipiens provides resistance to propoxur [24]. Because of the difference in the nature of carbamates and OPs, it is unlikely that the resistance mechanisms of HQT BfAChE and G247S Culex pipiens AChE are similar. In fact, our results show that HQT BfAChE is able to hydrolyse OPs. Therefore, the resistance we observed is a combination of both altered binding of the OPs and the ability to hydrolyse them. There is a slight activation of ATC hydrolysis by HQT BfAChE at concentrations of paraoxon above 1 mM. A transient activation of human AChE with nanomolar concentration of paraoxon has also been observed by Rosenfeld and Sultatos (personal communication). The difference in effective concentration range for paraoxon could be explained by the decreased catalytic efficiency of the HQT mutant. It appears that interaction of some OPs with AChE is more complex than currently accepted. Indeed, it has been proposed that a peripheral binding site could be involved in the interactions between AChE and certain OPs such as paraoxon [25]. The HQT mutant of BfAChE is the first AChE mutant capable of hydrolysing OPs. However, its OP hydrolase activity is 1–2 orders of magnitude lower compared to G117H hBChE, depending on the OP tested. The lack of efficiency of HQT BfAChE is probably related to the alteration of the active site structure. 4.3. Slow-binding behaviour with DFP Generally, equilibria between enzyme and reactants are set up rapidly, in the millisecond time scale. Kinetic measurements indicated that binding of DFP to HQT BfAChE involves a slow step. The behaviour of DFP was described by a mechanism, wherein there is an initial rapid interaction between the enzyme (E) and DFP (Y) to form an enzyme–DFP complex (EY), which then slowly isomerizes to EY*. Direct measurement of the hydrolysis of DFP using a specific fluoride electrode showed

T. Poyot et al. / Biochimica et Biophysica Acta 1764 (2006) 1470–1478

k1 E+S + Y k -3

k2 ES

E + P1

k -1

k3 k4 EY

EY*

k5

E + P2

k -4 Slow steps Scheme 2.

that EY* was further processed into E + P2, (P2 = F−). This leads to the following modification of the initial slow-binding model: We used the assumptions (k1, k− 1, k2, k3 and k− 3 >> k4, k− 4 and k5) and the same derivation method to derive the rate equations for Scheme 2 that we used to derive the equations for Scheme 1. The resulting expression for the time dependence of product (P1) formation is actually identical to the one derived from Scheme 1 (Eq. (2)). vi is unchanged in the new model (Eq. (3)). vss and k are affected because the rate of disappearance of EY* becomes (k− 4 + k5) according to Scheme 2 while it was k− 4 according to Scheme 1. This makes KEY = (k− 4 + k5)/k4. However, vss and k remain unchanged when expressed as a function of KEY (Eqs. (4) and (5)). Scheme 2 predicts that there should be a slow approach to the steady state of hydrolysis of DFP. However, due to slow equilibration of the fluoride electrode during the first 5 min of the assay, measurement of fluoride release does not allow detection of a lag in progress curve. Therefore, in Scheme 2, EY* corresponds to the phosphorylated enzyme. Phosphorylation of cholinesterases is generally considered to be irreversible, i.e. the phospho-enzyme does not return to organophosphate and enzyme. This corresponds to saying that k− 4 = 0. Then, it follows that the rate of disappearance of EY*, (k− 4 + k5), is reduced to k5, the dephosphorylation rate constant. This change will not affect the values for KY = 10.9 ± 2.7 mM and k4 = 1.80 ± 0.24 min− 1, however the product of KEY and k4 becomes k5 (instead of k− 4) the dephosphorylation rate (k5 = 0.080± 0.034 min− 1). 5. Conclusion This study describes the catalytic properties of a mutant of BfAChE with marginal OPase activity. This mutant was designed to reproduce the features of the first cholinesterase mutant with OPase activity, G117H hBChE. The HQT BfAChE mutant was resistant to OPs inhibition due to low affinity and presented an overall decreased catalytic and OP hydrolase efficiency compared to the G117H mutant of hBChE. Additionally, the G122H/Y124Q/S125T (HQT) mutant displayed Michaelis–Menten behaviour, i.e. it lost inhibition by excess substrate. To date, rational site-directed mutagenesis using molecular modelling of G117H-based mutants failed to generate an AChE mutant with sufficient OPase activity to be of therapeutic interest. However, a different rational approach

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using quantum mechanics/molecular mechanics may be more productive. This approach gave remarkable results when used to redesign hBChE to improve hydrolysis of (−)-cocaine [26]. Transition state simulations were used as the basis for the new mutant. In this study, the catalytic efficiency of hBChE for (−)cocaine was increased by more than 400-fold, by introducing 4 mutations to stabilize the transition state of hydrolysis, as predicted by molecular dynamics. Starting from a sound mechanistic model for hydrolysis of OPs by G117H-based OPase, this approach could be used for the design of a future generation of ChE mutants. Directed evolution may also represent an alternative method for generating ChE mutants capable of hydrolysing OPs at higher rates. However, this method relies on expression of functional AChE and BChE in E. coli, which has yet to be achieved [27, 28]. Acknowledgement This work was supported by DGA/DSP/STTC contract PEA 010807 (03co010–05) to PM and OL. References [1] B. Ballantyne, T.C. Marrs, Clinical and experimental toxicology of organophosphates and carbamates, Butterworth-Heinemann Publishers, Oxford, 1992. [2] D.E. Lenz, D.M. Maxwell, I. Koplowitz, C.R. Clark, B.R. Capacio, D.M. Cerasoli, J.M. Federko, C. Luo, A. Saxena, B.P. Doctor, C. Olson, Protection against soman or VX poisoning by human butyrylcholinesterase in guinea pigs and cynomolgus monkeys, Chem. Biol. Interact. 157–158 (2005) 205–210. [3] P. Masson, D. Josse, O. Lockridge, N. Viguié, C. Taupin, C. Buhler, Enzymes hydrolyzing organophosphates as potential scavengers against organophosphate poisoning, J. Physiol. (Paris) 92 (1998) 357–362. [4] Y. Ashani, S. Pistinner, Estimation of the upper limit of human butyrylcholinesterase dose required for protection against organophosphate toxicity: a mathematical based toxicokinetic model, Toxicol. Sci. 77 (2004) 358–367. [5] C.B. Millard, O. Lockridge, C.A. Broomfield, Design and expression of organophosphorus acid anhydride hydrolase activity in human butyrylcholinesterase, Biochemistry 34 (1995) 15925–15933. [6] O. Lockridge, R.M. Blong, P. Masson, M.T. Froment, C.B. Millard, C.A. Broomfield, A single amino acid substitution, Gly117His, confers phosphotriesterase (organophosphorus acid anhydride hydrolase) activity on human butyrylcholinesterase, Biochemistry 36 (1997) 786–795. [7] C.B. Millard, O. Lockridge, C.A. Broomfield, Organophosphorus acid anhydride hydrolase activity in human butyrylcholinesterase: synergy results in a somanase, Biochemistry 37 (1998) 237–247. [8] L.M. Schopfer, A. Ticu Boeck, C.A. Broomfield, O. Lockridge, Mutants of human butyrylcholinesterase with organophosphate hydrolase activity; evidence that His117 is a general base catalyst for hydrolysis of echothiophate, J. Med. Chem. Def. 2 (2004) 1–21. [9] M. Harel, J.L. Sussman, E. Krejci, S. Bon, P. Chanal, J. Massoulié, I. Silman, Conversion of acetylcholinesterase to butyrylcholinesterase: modeling and mutagenesis, Proc. Natl. Acad. Sci. U. S. A. 89 (1992) 10827–10831. [10] Z. Radic, N.A. Pickering, D.C. Vellom, S. Camp, P. Taylor, Three distinct domains in the cholinesterase molecule confer selectivity for acetyl- and butyrylcholinesterases inhibitors, Biochemistry 34 (1993) 12074–12084. [11] Y. Nicolet, O. Lockridge, P. Masson, J.C. Fontecilla-Camps, F. Nachon, Crystal structure of human butyrylcholinesterase and of its complexes with substrate and products, J. Biol. Chem. 278 (2003) 41141–41147. [12] R.D. Newcomb, P.M. Campbell, D.L. Ollis, E. Cheah, R.J. Russell, J.G.

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