Protein Engineering vol. 16 no. 7 pp. 463-465, 2003
© 2003 Oxford University Press
A long insertion reverts the functional effect of a substitution in acetylcholinesterase
Institute for Technical Biochemistry, Allmandring 31, University of Stuttgart, 70569 Stuttgart, Germany
1 To whom correspondence should be addressed. e-mail: itbfvi{at}po.uni-stuttgart.de
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Abstract |
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Proteins are thought to undertake single substitutions, deletions and insertions to explore the fitness landscape. Nevertheless, the ways in which these different kind of mutations act together to alter a protein phenotype remain poorly described. We introduced incrementally the single substitution W290A and a 26 amino acid long insertion at the 297 location in the Nippostrongylus brasiliensis acetylcholinesterase B sequence and analysed in vitro the induced changes in the hydrolysis rate of three hemi-substrates: pirimicarb, paraoxon methyl and omethoate. The substitution decreased the hydrolysis rate of the three hemi-substrates. The insertion did not influence this kinetic alteration induced by the substitution for the former hemi-substrate, but reverted it for the two others. These results show that two different kinds of mutations can interact together to influence the direction of a protein’s adaptative walk on the fitness landscape.
Keywords: cholinesterase/fitness landscape/insertion/mutation/protein evolution
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Mutations are the basis of evolution. Single amino acid substitutions, deletions and insertions are thought together to alter proteins to modulate their shape and evolve organisms. The exploration of the fitness landscape by a given protein is expected to depend directly on the number and kind of incremental changes that its structure undertakes. Nevertheless, the outcome of the interactions of these changes on the direction of a protein’s adaptive walk is surprisingly poorly documented (Beier et al., 2000
; Qvarnström and Swedberg, 2000). How can a substitution and an insertion interact together to influence this walk?
Acetylcholinesterase (AChE) is a key enzyme in the nervous system. It is responsible for the hydrolysis of the neurotransmitter acetylcholine in the synapse, but is also able to hydrolyse other substrates or hemi-substrates such as phosphate esters or carbamates. Its high turnover rate of 25 000 s–1, and the possibility of easily following its activity with a simple spectrophotometric method (Ellman et al., 1961), contributed to the popularity of AChE as a model enzyme and make it very suitable for addressing evolution questions. Among the different sub-sites in the enzyme, the so-called ‘acyl pocket’ was shown to stabilize the substrate in the active site by forming a clamp around its acyl part (Sussman et al., 1991; Harel et al., 1992) and alterations in this sub-site were reported to change ligand binding in different cholinesterases (Harel et al., 1992; Villatte et al., 2000a,b).
In this paper, we show that the biochemical phenotype of acetylcholinesterase induced by a residue substitution can be reverted by a 26 amino acid insertion at a different location in the sequence. These results illustrate the link between the incremental occurrence of different kinds of mutations that could occur in evolving enzymes and their functionality leading to back steps on the fitness landscape.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Chemicals
Chemicals were purchased from Sigma Aldrich (Germany) if not indicated otherwise.
Mutagenesis and enzyme production
All enzymes were expressed in a Pichia pastoris X33 strain transformed with a pPICalphaB vector bearing the Nippostrongylus brasiliensis AChE B gene under the control of a methanol-inducible promoter, according to Hussein et al. (Hussein et al., 1999). After protein induction, cultures were centrifuged at 12 000 r.p.m. for 5 min and the supernatant was used as the source of enzyme. Mutagenesis was performed by primer extension (QuikChange kit, Stratagene) following the manufacturer’s instructions and the sequence was checked by DNA sequencing.
Kinetics
The determination of the AChE activity by spectrophotometry was performed according to Ellman et al. (Ellman et al., 1961) by recording the absorbance at 412 nm on a Pharmacia-Biochrom 4060 spectrophotometer in 25 mM phosphate buffer pH 7 containing 1 mM acetylthiocholine and 3x10–4 5,5'-dithiobis(2-nitrobenzoic acid) (1 ml final volume) at 25°C.
The determination of bimolecular rate constants (ki) was performed according to Aldridge (Aldridge, 1950) and following the reaction scheme
where E = enzyme, CX = carbamate or organophosphate compound, X = leaving group and C = remaining group. To estimate the bimolecular rate constants (ki), the enzymes were incubated for various times with different insecticide (Riedel-de Haën) concentrations with [CX] at least 10-fold higher than [E] in 25 mM phosphate buffer pH 7 (1 mg/ml BSA) at 25°C. The change in the concentration of the free enzyme [E] over time was estimated by recording the remaining activity measured with 1 mM acetylthiocholine as substrate. This variation followed pseudo-first-order kinetics, ln[E]/[E0] = –ki[CX]t (where t represents the time of incubation, [CX] the inhibitor concentration, [E0] the initial enzyme concentration and [E] the free enzyme concentration after incubation with inhibitor) since all graphs obtained were linear. This suggests that dephosphorylation or decarbamylation (k3) remained negligible over the duration of the experiment (5 min), as already observed by various authors (Rush et al., 1986; Mutero et al., 1992; Villatte et al., 1998). Plots of ln(% AChE remaining activity) against time were drawn and the slope divided by the inhibitor concentration gave the bimolecular rate constant (ki).
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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We used the N.brasiliensis AChE B as a model enzyme to analyse the kinetic changes induced by a substitution and an insertion in the protein sequence. We replaced the tryptophan at location 290 [numbering according to Torpedo californica AChE (Sussman et al., 1991
)] with an alanine and introduced a 26 amino acid long insertion (EMLPMEAPFGPITEM LPMEAPFGPIT) at location 297 (Figure 1) in a second experiment. According to the structure of the Torpedo enzyme (Sussman et al., 1991), the substitution took place in the so-called ‘acyl pocket’ of the enzyme, responsible for the accommodation of the ester part of the substrate (Harel et al., 1992). Amino acid replacements in the acyl pocket of the Drosophila cholinesterase led to alterations in the hydrolysis of acetylthiocholine and various carbamates and organophosphates used as insecticides (Villatte et al., 2000a). The insertion was randomly performed in a loop exposed to the solvent, at the root of an 
-helix. Then, the ability of the wild-type enzyme and the mutants to hydrolyse the carbamate pirimicarb and the phosphate esters paraoxon methyl and omethoate was investigated. The three molecules (Figure 2) are used as insecticides and contain ester bonds that are hydrolysed by AChE, even if the decarbamylation and the dephosphorylation rates of the enzyme are low. The bimolecular rate constant ki is proportional to the hydrolysis rate of the hemi-substrates. It appeared that the single mutation, W290A, led to a reduced hydrolysis rate for all three substrates (Figure 3). The replacement of the tryptophan with an alanine at location 290 may lead to a decreased stabilization of the substrate molecule in the active site compared with the wild-type enzyme, because of the lower steric hindrance of the alanine lateral chain. Then, three different effects of the insertion at the location 297 were observed, compared with the substitution alone: no change for pirimicarb, an increase in the hydrolysis rate of paraoxon-methyl and a restoration of the hydrolysis rate of omethoate to a higher rate than the wild-type (Figure 3). It can be assumed that the structural deformation of the already substituted acyl pocket induced by the insertion increased the stabilization of the paraoxon methyl and the omethoate but not the pirimicarb. As reported elsewhere (Villatte et al., 2000a), the structure of the hemi-substrates is different and may influence significantly the position of the molecule in the active site and thus the hydrolysis rate. It is interesting that the enzyme function is not disrupted even with this long insertion, probably exposed to the solvent despite its hydrophobic properties.
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Using a two-dimensional lattice thermodynamic model, Xia and Levitt (Xia and Levitt, 2002
) recently proposed a solution to the ‘Levinthal paradox’ according to which a protein surprisingly finds the optimal sequence in a short time in a flat fitness landscape: the nearly infinite possibilities of changes offered to the sequence could be faced by a high recombination/mutation rate ratio. This result underscores the need for different molecular events to occur simultaneously during protein evolution. The biochemical effect resulting from these interactions remains poorly described, especially for enzymes, which play a fundamental role in life. One of the main evolutionary drivers in enzyme evolution is their catalytic properties. The data presented here demonstrate first that the plasticity of an enzyme can be high enough to tolerate a long insertion in the sequence without disruption of the catalytic function. Second, the results show that the kinetic effects led by a single substitution can be reverted to different extents by an insertion, depending on the substrate. This could be an example of a mechanism leading to a reverse geared step in the adaptive walk of a protein on the fitness landscape. |
Acknowledgement |
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The authors thank Dr A.Hussein (UK) for the kind gift of the N.brasilisensis AChE B gene.
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References |
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Received January 28, 2003; revised April 25, 2003; accepted June 6, 2003.
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