Improvement of Drosophila acetylcholinesterase stability by elimination of a free cysteine.
BACKGROUND
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.


RESULTS
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.


CONCLUSIONS
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.
Fremaux
Isabelle
I
Laboratoire de Synthèse et Physicochimie des Molécules d'Intérêt Biologique, UMR 5068, Université Paul Sabatier, 31062, Toulouse, France. isa10yann@yahoo.fr
Mazères
Serge
S
Brisson-Lougarre
Andrée
A
Arnaud
Muriel
M
Ladurantie
Caroline
C
Fournier
Didier
D
D000110:Acetylcholinesterase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D004331:Drosophila melanogaster / Q000201:enzymology*; D004795:Enzyme Stability; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D016296:Mutagenesis; D011489:Protein Denaturation; D014633:Valine / Q000235:genetics
D000110:Acetylcholinesterase; D014633:Valine; D003545:Cysteine