Acetylcholinesterase (AChE, EC 3.1.1.7) 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.[9] 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].

There are eight cysteines in the wild type cholinesterase [17], six are involved in intrachain disulfide bonds, one is involved in an interchain disulfide bond and one, at position 290 (328 with the precursor numbering) remains free [18]. This free cysteine is half buried in the protein, located in a loop near the disulfide bond formed by cysteines 292 and 307 (Fig. 1 and 2) [19]. When comparing the primary sequence of cholinesterases, it appears that this cysteine is not conserved and thus is not essential for the function of the enzyme. In the present study, we used mutagenesis to change this free cysteine for a hydrophobic residue with a similar size, a valine to enhance the stability of the enzyme.

Thermodynamic and thermal (also called long-term) stabilities are usually closely related since thermal denaturation is considered to be a two-step process involving a reversibly unfolded intermediate [20]. But the two stabilities do not necessarily correlate due to independent unfolding of microdomains [21,22]. Therefore, several denaturation methods were applied. In all situations analyzed, denaturation of AChE was irreversible and followed apparent first order kinetics. Stability was then characterized by the half-life (t50), the time at which 50% of an initial enzymatic activity is preserved. The effect of freezing was measured from the recovery after one freeze-thaw cycle (Table 1).

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 [7]. 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 [30] 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 [34], it can sometimes lead to loss of biological activity [35]. 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 [36]. To test the relevance of this phenomenon for stability, we used the "foot printing system" of Tullius and Dombrowski [37] using the EDTA complex of iron(II) to generate hydroxyl radical form hydrogen peroxide as previously performed with AChE [38]. 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.

A.B-L performed the in vitro mutagenesis experiment, I.F. and M.A. produced and purified the proteins. S.M. and C.L. carried out the stability measurements, D.F. designed the study. All authors read and approved the final manuscript.