Improvement of Drosophila acetylcholinesterase stability by elimination of a free cysteine
© Fremaux et al; licensee BioMed Central Ltd. 2002
Received: 18 May 2002
Accepted: 30 July 2002
Published: 30 July 2002
Acetylcholinesterase is irreversibly inhibited by organophosphate and carbamate insecticides allowing its use for residue detection with biosensors. Drosophila acetylcholinesterase is the most sensitive enzyme known and has been improved by in vitro mutagenesis. However, it is not sufficiently stable for extensive utilization. It is a homodimer in which both subunits contain 8 cysteine residues. Six are involved in conserved intramolecular disulfide bridges and one is involved in an interchain disulfide bridge. The 8th cysteine is not conserved and is present at position 290 as a free thiol pointing toward the center of the protein.
The free cysteine has been mutated to valine and the resulting protein has been assayed for stability using various denaturing agents: temperature, urea, acetonitrile, freezing, proteases and spontaneous-denaturation at room temperature. It was found that the C290V mutation rendered the protein 1.1 to 2.7 fold more stable depending on the denaturing agent.
It seems that stabilization resulting from the cysteine to valine mutation originates from a decrease of thiol-disulfide interchanges and from an increase in the hydrophobicity of the buried side chain.
Acetylcholinesterase (AChE, EC 18.104.22.168) is a serine hydrolase, which catalyzes the hydrolysis of acetylcholine. This enzyme is the target of organophosphate and carbamate insecticides which phosphorylate or carbamoylate the serine of the active site blocking the hydrolysis of the neurotransmitter acetylcholine. The post-synaptic membrane then remains depolarised and synaptic transmission cannot take place so the insect dies. These compounds are used to control various agricultural pests: insects, acari and nematodes. One of the consequences is that pesticide residues remain in the environment and are potentially toxic for all animals including humans since cholinergic transmission is well conserved.
Insecticide residues can be detected with biosensors using AChE as biological element. Such a method has been described to detect low levels of contaminants in crops, soil, water or food samples using various kinds of biosensors [1, 2]. Drosophila AChE was found to be the most sensitive enzyme when compared to enzymes of non-insect origin and in-vitro-mutagenesis was used to select enzymes up to 300-fold more sensitive [3, 4]. But like most enzymes from mesophilic organisms, Drosophila AChE is not stable, and this instability precludes its utilization in biosensors. It can be stabilized by additives: protein such as bovine serum albumin, reversible inhibitor or polyethylene glycol. Alternatively, stabilization can also be achieved by encapsulation in liposomes [5–8]. Another way to stabilize the enzyme is to use in vitro mutagenesis to modify the primary structure of the protein. This method has the advantage of stabilizing the enzyme during its synthesis leading to higher production and higher purification yields. Several kinds of mutation may stabilize a protein, among them there is elimination of free sulfhydryl groups.
Wang et al. suggested that removal of nonessential free cysteines by serine could be a general method for preparing muteins with higher stability, specific activity and homogeneity. Subsequent assays have shown that mutation of free cysteines has been found to increase the half-life of proteins [10–14]. However, some exemples have been reported where elimination of a free cysteine did not improve the stability of protein and even decreased it [6, 15, 16].
Results and discussion
Stability of wild type and mutated AChE (mean ± SEM) and an index of the increase in stability i.e. the ratio between the measurement made for the mutant and the wild type enzymes.
t50 at 45°C in min.
17.4 ± 2.1 (n = 10)
24.1 ± 4.3 (n = 5)
t50 in 4 M urea in min.
12.5 ± 1.2 (n = 7)
34.2 ± 2.0 (n = 5)
Recovery after freezing
0.24 ± 0.04 (n = 8)
0.49 ± 0.04 (n = 7)
t50 at room temperature in days
1.5 ± 0.2 (n = 5)
3.3 ± 0.5 (n = 4)
t50 in 20% acetonitrile in min.
0.94 ± 0.1 (n = 12)
1.4 ± 0.4 (n = 5)
t50 in presence of 0.1 mg/ml pronase
7.9 ± 0.2 (n = 9)
10.0 ± 0.6 (n = 9)
t50 in H2O2 in min.
4.6 ± 0.5 (n = 6)
4.0 ± 0.4 (n = 6)
We first analyzed denaturation with the most common methods used to study protein denaturation: incubation at high temperature or in the presence of urea. Incubation of the wild type enzyme at 45°C resulted in the progressive irreversible denaturation of the enzyme with a 17 min half-life. Mutation of cysteine 290 to valine increased this half-life to 24 min. It also provided resistance to urea denaturation: the mutated enzyme was 2.7-fold more stable than the wild type if we consider the ratio of half-life time in 4 M urea (Table 1).
Lyophilisation is the most efficient method to store Drosophila AChE, however freezing denatures the enzyme at dilutions below the concentration of 1 mmole per liter . We thus checked if the C290V mutation protects the enzyme at the concentration of 10 nM. Following one freezing to -20°C, 75% of the wild type enzyme was denatured while only 50% of the mutated enzyme activity was lost.
The utilization of AChE in a biosensor can last several days in some continuous flow devices. In fact, as the inhibition is irreversible, the longer AChE is incubated with the sample, the lower the detection limit of the biosensor. However, the enzyme activity has to decrease from inhibition and not from spontaneous denaturation of the protein. We thus incubated the wild type and the mutated enzyme at 20°C. Half the enzyme was denatured in 1.5 days; the mutation doubled this half life.
Detection of insecticides in food requires their extraction with organic solvent. Although the solvent should be eliminated before the assay, low amounts may remain in solution and inactivate the enzyme. We used acetonitrile as model because it is soluble in water. This organic solvent strongly denatured the protein which had a half-life in 20% acetonitrile of 1.3 minutes. This half-life was slightly increased to 1.4 minutes by the mutation.
As some proteases may be present in the sample to be assayed for pesticide residues, we tested the effect of the mutation on the proteolytic sensitivity of the enzyme. Actually, it has been reported that a protein's resistance to proteolysis increases with its conformational stability [23, 24] due to the fact that the susceptibily to proteolysis reflects the rate of local unfolding [25, 26]. Our data support this relation between stability and proteolytic susceptibility since the mutated enzyme was 1.3 more resistant to proteolytic attack than the wild type enzyme in the experimental conditions used.
Several hypotheses can be put forward to explain this increased stability. A first hypothesis involves the deletion of the highly reactive sulfhydryl group which could interact with the neighboring disulfide bond. Thiol-disulfide interchanges have been reported in proteins [27–29] including acetylcholinesterase  and changes involving a free sulfhydryl group and a disulfide bond have been found to result in the irreversible denaturation of proteins [31–33]. As the disulfide exchange reaction originates from nucleophilic attack on a sulfur atom of the disulfide, the proximity of the disulfide bond formed by cysteines 292 and 307 enforces this hypothesis. The inter Cβ distance between Cys 290 and Cys 292 or Cys 307 are 5 Å or 7.7 Å respectively (Fig. 1). Actually, mutation of cysteine 292 to valine resulted in an inactive protein suggesting that disruption of the bond between cysteines 292 and 307 results in misfolding since the loop is not involved in the structure of the active site.
A free sulfhydryl group may also mediate the formation of dimer and oligomer. Although oligomerization usually has a stabilizing effect , it can sometimes lead to loss of biological activity . As the side chain of cysteine 290 points towards the interior of the protein and is inaccessible to the solvent, it is unlikely that this cysteine is responsible for the formation of tetramers.
Air oxidation of thiols is an important source of thermal inactivation of lysozyme . To test the relevance of this phenomenon for stability, we used the "foot printing system" of Tullius and Dombrowski  using the EDTA complex of iron(II) to generate hydroxyl radical form hydrogen peroxide as previously performed with AChE . The C290V mutation did not increase the half life of the protein. Thus, stabilization does not seem to originate from the elimination of an oxidation site.
The contribution of individual amino acids to the stability of a specific protein has often been found to be difficult to assess since the elimination of one type of interaction often affects other types of interactions. This can happen in our mutation: by replacing a cysteine by a valine, we not only eliminated a reactive thiol, we also increased the hydrophobicity of the side chain which is oriented towards the heart of the protein (Fig. 2). As increasing the hydrophobicity of buried residues usually increases the stabilityof proteins [39–42], we may hypothesize that this rule applied here.
Thus, removal of the free cysteine 290 most probably stabilized Drosophila AChE by two mechanisms: inhibition of disulfide exchange and increase of the hydrophobicity of the side chain.
Protein production and purification
cDNA encoding Drosophila melanogaster AChE and mutant were expressed with the baculovirus system . We expressed a soluble dimeric form deleted from a hydrophobic peptide at the C-terminal end with a 3 × histidine tag replacing the loop from amino-acids 103 to 136. This external loop is at the other end of the molecule with respect to the active and its deletion does not affect either the activity or the stability of the enzyme. Secreted AChE was purified to homogeneity using the following steps, ammonium sulfate precipitation, ultrafiltration with a 50 kDa cut off membrane, affinity chromatography with procainamide as ligand, NTA-nickel chromatography and anion exchange chromatography . Activity was recorded at 25°C in 25 mM phosphate buffer pH 7, with 1 mM acetylthiocholine iodide as substrate using the method of Ellman et al. . Residue numbering followed that of the mature protein.
All denaturation experiments were performed with 10 picomoles enzyme in 1 ml 25 mM phosphate buffer pH7 at 25°C. AChE was incubated in denaturing conditions and the remaining activity was measured by taking aliquots at regular times since residual enzymatic activity is dependent on the time of incubation. To analyze thermosensitivity, enzymes were incubated at 45°C instead of 25°C and 1 mg/ml Bovine Serum Albumin was added to the buffer. Aliquots were mixed with cold buffer chilled on ice and the solution was incubated at 25°C for ten minutes before recording the remaining activity. For urea denaturation, unfolding of AChE was induced by adding 4 M urea to the incubation buffer. Aliquots were diluted 10-fold in 25 mM phosphate buffer pH7 before measuring the remaining activity. The effect of freezing was estimated by measuring the remaining activity after one day at -20°C. Stability at room temperature was measured by recording the activity after incubation at 20°C. The effect of organic solvent was followed by incubation of the enzyme in 20% acetonitrile. The effect of protease sensitivity was determined by incubation of AChE with 0.1 mg/ml pronase. To follow the effect of oxygen radical on denaturation 10 mM H2O2, 1 mM FeCl3, 2 mM EDTA and 40 mM ascorbic acid were added to the incubation medium following the procedure of Weiner et al. .
This research was supported by grants from CEE (AchEB QLK3-CT-2000-00650, and Safegard QLK3-CT-2000-000481).
- Marty JL, Sode K, Karube I: Biosensor for detection of organophosphate and carbamate insecticides. Electroanalysis. 1992, 4: 801-803.View ArticleGoogle Scholar
- Bachmann TT, Leca B, Vilatte F, Marty J-L, Fournier D, Schmid R: Improved multianalyte detection of organophosphates and carbamates with disposable multielectrode biosensors using recombinant mutants of Drosophila acetylcholinesterase and artificial neural networks. Biosens Bioelectron. 2000, 15: 193-201. 10.1016/S0956-5663(00)00055-5.PubMedView ArticleGoogle Scholar
- Villatte F, Marcel V, Estrada-Mondaca S, Fournier D: Engineering sensitive acetylcholinesterase for detection of organophosphate and carbamate insecticides. Biosens Bioelectron. 1998, 13: 157-162. 10.1016/S0956-5663(97)00108-5.PubMedView ArticleGoogle Scholar
- Boublik Y, Saint-Aguet P, Lougarre A, Arnaud M, Villatte F, Estrada-Mondaca S, Fournier D: Acetylcholinesterase engineering for detection of insecticide residues. Protein Eng. 2002, 15: 43-50. 10.1093/protein/15.1.43.PubMedView ArticleGoogle Scholar
- Payne CS, Saeed M, Wolfe AD: Ligand stabilization of cholinesterases. Biochim Biophys Acta. 1989, 999: 46-51. 10.1016/0167-4838(89)90028-9.PubMedView ArticleGoogle Scholar
- Wilson EJ, Massoulié J, Bon S, Rosenberry TL: The rate of thermal inactivation of Torpedo acetylcholinesterase is not reduced in the C231S mutant. FEBS Lett. 1996, 379: 161-164. 10.1016/0014-5793(95)01504-3.PubMedView ArticleGoogle Scholar
- Estrada-Mondaca S, Fournier D: Stabilization of recombinant Drosophila acetylcholinesterase. Protein. 1998, 12: 166-172. 10.1006/prep.1997.0831.Google Scholar
- Nasseau M, Boublik Y, Meier W, Winterhalter M, Fournier D: Substrate-permeable encapsulation of enzymes maintains effective activity, stabilizes against denaturation, and protects against proteolytic degradation. Biotechnol Bioeng. 2001, 75: 615-618. 10.1002/bit.10074.PubMedView ArticleGoogle Scholar
- Wang A, Lu S-D, Mark DF: Site-specific mutagenesis of the human interleukin-2 gene: structure-function analysis of the cysteine residues. Science. 1984, 224: 1431-1433.PubMedView ArticleGoogle Scholar
- McRee DE, Redford SM, Getzoff ED, Lepock JR, Hallewell RA, Tainer JA: Changes in crystallographic structure and thermostability of a Cu, Zn superoxide dismutase mutant resulting from the removal of a buried cysteine. J Biol. Chem. 1990, 265: 14234-14241.PubMedGoogle Scholar
- Faletto MB, Linko P, Goldstein JA: Single amino acid mutation (Ser180 -> Cys) determines the polymorphism in cytochrome p450g (P4502C13) by altering protein stability. J Biol Chem. 1992, 267: 2032-2037.PubMedGoogle Scholar
- Miyazaki Y, Matsufuji S, Murakami Y, Hayashi S: Single amino-acid replacement is responsible for the stabilization of ornithine decarboxylase in HMO A cells. Eur J Biochem. 1993, 214: 837-844.PubMedView ArticleGoogle Scholar
- Amaki Y, Nakato H, Yamane T: Role of cysteine residues in esterase from Bacillus stearothermophilus and increasing its thermostability by replacement of cysteines. Appl Microbiol Biotechnol. 1994, 40: 664-668. 10.1007/s002530050047.PubMedView ArticleGoogle Scholar
- Lett CM, Rosu-Myles MD, Frey HE, Guillemette JG: Rational design of a more stable yeast iso-1-cytochrome C. Biochim Biophys Acta. 1999, 1432: 40-48. 10.1016/S0167-4838(99)00071-0.PubMedView ArticleGoogle Scholar
- Eijsink VGH, Veltman OR, Aukema W, Vriend G, Venema G: Structural determinants of the stability of thermolysin-like proteinases. Nat Struct Biol. 1995, 2: 374-379.PubMedView ArticleGoogle Scholar
- Loyola C, Savedra C, Gómez I, Vásquez C: The amino acid substitution of cysteine 167 by serine (C167 S) in BstVI restriction endonuclease of Bacillus stearothermophilus V affects its conformation and thermostability. Biochimie. 1999, 81: 261-266. 10.1016/S0300-9084(99)80060-1.PubMedView ArticleGoogle Scholar
- Hall LCM, Spierer P: The Ace locus of Drosophila melanogaster: structural gene for acetylcholinesterase with an unusual 5' leader. EMBO J. 1986, 5: 2949-2954.PubMed CentralPubMedGoogle Scholar
- Mutero A, Fournier D: Post-translational modifications of Drosophila acetylcholinesterase. In vitro mutagenesis and expression in Xenopus oocytes. J Biol Chem. 1992, 267: 1695-1700.PubMedGoogle Scholar
- Harel M, Kryger G, Rosenberry TL, Mallender WD, Lewis T, Flechter RJ, Guss JM, Silman I, Sussman JL: Three dimensional structures of Drosophila melanogaster acetylcholinesterase and of its complexes with potent inhibitors. Protein Sci. 2000, 9: 1063-1072.PubMed CentralPubMedView ArticleGoogle Scholar
- Mozhaev VV: Mechanism-based strategies for protein thermostabilization. Trends Biotechnol. 1993, 11: 88-95.PubMedView ArticleGoogle Scholar
- Chrunysk BA, Wetzel R: Breakdown in the relationship between thermal and thermodynamic stability in an interleukin-1β point mutant modified in a surface loop. Protein Eng. 1993, 6: 733-738.View ArticleGoogle Scholar
- Arnone MI, Birolo L, Pascarella MV, Cubellis MV, Bossa F, Sannia G, Marino G: Stability of aspartate aminotransferase from Sulfolobus solfataricus. Protein Eng. 1997, 10: 237-248. 10.1093/protein/10.3.237.PubMedView ArticleGoogle Scholar
- Matsumura M, Yasumura S, Aiba S: Cumulative effect of intragenic amino-acid replacements on the thermostability of a protein. Nature. 1986, 323: 356-358.PubMedView ArticleGoogle Scholar
- Ogasahara K, Tunasawa S, Soda Y, Yutani K, Sugino Y: Effect of single amino acid substitutions on the protease susceptibility of tryptophan synthase alpha subunit. Eur J Biochem. 1985, 150: 17-21.PubMedView ArticleGoogle Scholar
- Zettlmeissl G, Rudolf R, Jaenicke R: Limited proteolysis to study the kinetics of protein folding: conformational rearrangements in acid-dissociated lactic dehydrogenase as determined by pepsin digestion. Arch Biochem Biophys. 1983, 224: 161-168.PubMedView ArticleGoogle Scholar
- Braxton S, Wells JA: Incorporation of a stabilizing Ca2+ binding loop into subtinilin BN'. Biochemistry. 1992, 31: 7796-7801.PubMedView ArticleGoogle Scholar
- Sogami M, Petersen HA, Foster JF: The microheterogeneity of plasma albumins. V. Permutations in disulfide pairings as probable source of microheterogeneity in bovine albumin. Biochemistry. 1969, 8: 49-58.PubMedView ArticleGoogle Scholar
- Nikkel HJ, Foster JF: A reversible sulfhydryl-catalyzed structural alteration of bovine mercaptalbumin. Biochemistry. 1971, 10: 4479-4486.PubMedView ArticleGoogle Scholar
- Zale SE, Klibanov AM: Why does ribonuclease irreversibly inactivate at high temperature. Biochemistry. 1986, 25: 5432-5444.PubMedView ArticleGoogle Scholar
- Eichler J, Kreimer DI, Varon L, Silman I, Weiner L: A "Molten globule" of Torpedo acetylcholinesterase undergoes thiol-disulfide exchange. J Biol Chem. 1994, 269: 30093-30096.PubMedGoogle Scholar
- McKenzie HA, Ralston GB, Shaw DC: Location of sulfhydryl and disulfide groups in bovine β-lactoglobulins and effects of urea. Biochemistry. 1972, 11: 4539-4546.PubMedView ArticleGoogle Scholar
- Perry LJ, Wetzel R: Unpaired cysteine-54 interferes with the ability of an engineered disulfide to stabilize T4 lysozyme. Biochemistry. 1986, 25: 733-739.PubMedView ArticleGoogle Scholar
- Bonaccorsi di Patti MC, Carri MT, Gabbianelli R, Da Gai R, Volpe C, Giartosio A, Rotilio G, Battistoni A: A free cysteine residue at the dimer interface decreases conformational stability of Xenopus laevis copper, zinc superoxide dismutase. Arch Biochem Biophys. 2000, 377: 284-289. 10.1006/abbi.2000.1786.PubMedView ArticleGoogle Scholar
- Takagi H, Hirai K, Wada M, Nakamori S: Enhanced thermostability of the single-Cys mutant subtilisin E under oxidizing conditions. J Biochem. 2000, 128: 585-589.PubMedView ArticleGoogle Scholar
- Mark DF, Lu SD, Creasey AA, Yamamoto R, Lin LS: Site-specific mutagenesis of the human fibroblast interferon gene. Proc Natl Acad Sci USA. 1984, 81: 5662-5666.PubMed CentralPubMedView ArticleGoogle Scholar
- Perry LJ, Wetzel R: The role of cysteine oxidation in the thermal inactivation of T4 lysozyme. Protein. 1987, 1: 101-105.View ArticleGoogle Scholar
- Tullius TD, Dombroski BA: Hydroxyl radical "footprinting": high-resolution information about DNA-protein contacts and application to λ repressor and Cro protein. Proc Natl Acad Sci USA. 1986, 83: 5469-5473.PubMed CentralPubMedView ArticleGoogle Scholar
- Weiner L, Kreimer D, Roth E, Silman I: Oxidative stress transforms acetylcholinesterase to a molten-globule-like state. Biochem Biophys Res Com. 1994, 198: 915-922. 10.1006/bbrc.1994.1130.PubMedView ArticleGoogle Scholar
- Yutani K, Ogasahara K, Sugino Y, Matsushiro A: Effect of a single amino acid substitution on stability of conformation of a protein. Nature. 1977, 267: 274-275.PubMedView ArticleGoogle Scholar
- Yutani K, Ogasahara K, Aoki K, Kakuno T, Sugino Y: Effect of amino acid residues on conformational stability in eight mutant proteins variously substitued at a unique position of the tryptophan synthase α-subunit. J Biol Chem. 1984, 259: 14076-14081.PubMedGoogle Scholar
- Yutani K, Ogasahara K, Tsujita T, Sugino Y: Dependence of conformational stability on hydrophobicity of the amino acid residue in a series of variant proteins substituted at a unique position of the tryptophan synthase α subunit. Proc Natl Acad Sci USA. 1987, 84: 4441-4444.PubMed CentralPubMedView ArticleGoogle Scholar
- Matsumura M, Becktel WJ, Mattews BW: Hydrophobic stabilization in T4 lysozyme determined directly by multiple substitution of Ile 3. Nature. 1988, 334: 406-410. 10.1038/334406a0.PubMedView ArticleGoogle Scholar
- Chaabihi H, Fournier D, Fedon Y, Bossy JP, Ravallec M, Devauchelle G, Cérutti M: Biochemical characterization of Drosophila melanogaster acetylcholinesterase expressed by recombinant baculovirus. Biochem Biophys Res Comm. 1994, 203: 734-742. 10.1006/bbrc.1994.2243.PubMedView ArticleGoogle Scholar
- Ellman GL, Courtney KD, Andres KD, Featherstone RM: A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmac. 1961, 7: 88-95. 10.1016/0006-2952(61)90145-9.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.