Fremaux Isabelle, Mazères Serge, Brisson-Lougarre Andrée, Arnaud Muriel, Ladurantie Caroline, Fournier Didier
Laboratoire de Synthèse et Physicochimie des Molécules d'Intérêt Biologique, UMR 5068, Université Paul Sabatier, 31062, Toulouse, France.
BMC Biochem. 2002 Jul 30;3:21. doi: 10.1186/1471-2091-3-21.
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.
有机磷酸酯类和氨基甲酸酯类杀虫剂可不可逆地抑制乙酰胆碱酯酶，从而使其可用于生物传感器进行残留检测。果蝇乙酰胆碱酯酶是已知最敏感的酶，并且已通过体外诱变得到改进。然而，它的稳定性不足以支持广泛应用。它是一种同二聚体，两个亚基均含有8个半胱氨酸残基。其中6个参与保守的分子内二硫键，1个参与链间二硫键。第8个半胱氨酸不保守，位于290位，作为游离巯基指向蛋白质中心。
将游离半胱氨酸突变为缬氨酸，并使用各种变性剂对所得蛋白质的稳定性进行了测定：温度、尿素、乙腈、冷冻、蛋白酶以及室温下的自发变性。结果发现，根据变性剂的不同，C290V突变使蛋白质的稳定性提高了1.1至2.7倍。
半胱氨酸突变为缬氨酸所导致的稳定性提高似乎源于硫醇-二硫键交换的减少以及埋藏侧链疏水性的增加。
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