Cléry-Barraud Cécile, Ordentlich Arie, Grosfeld Haim, Shafferman Avigdor, Masson Patrick
Centre de Recherches du Service de Santé des Armées, Unité d'enzymologie, France.
Eur J Biochem. 2002 Sep;269(17):4297-307. doi: 10.1046/j.1432-1033.2002.03122.x.
The effects of pressure on structure and activity of recombinant human acetylcholinesterase (rHuAChE) were investigated up to a pressure of 300 MPa using gel electrophoresis under elevated hydrostatic pressure, fluorescence of bound 8-anilinonaphthalene-1-sulfonate (ANS) and activity measurements following exposure to high pressure. Study of wild-type enzyme and three single mutants (D74N, E202Q, E450A) and one sextuple mutant (E84Q/E292A/D349N/E358Q/E389Q/D390N) showed that pressure exerts a differential action on wild-type rHuAChE and its mutants, allowing estimation of the contribution of carboxylic amino acid side-chains to enzyme stability. Mutation of negatively charged residues D74 and E202 by polar side-chains strengthened heat or pressure stability. The mutation E450A and the sextuple mutation caused destabilization of the enzyme to pressure. Thermal inactivation data on mutants showed that all of them were stabilized against temperature. In conclusion, pressure and thermal stability of mutants provided evidence that the residue E202 is a determinant of structural and functional stability of HuAChE.
利用高压凝胶电泳、结合的8-苯胺基萘-1-磺酸盐（ANS）荧光以及高压处理后的活性测量，研究了高达300 MPa压力对重组人乙酰胆碱酯酶（rHuAChE）结构和活性的影响。对野生型酶和三个单突变体（D74N、E202Q、E450A）以及一个六重突变体（E84Q/E292A/D349N/E358Q/E389Q/D390N）的研究表明，压力对野生型rHuAChE及其突变体具有不同的作用，从而能够评估羧基氨基酸侧链对酶稳定性的贡献。用极性侧链取代带负电荷的残基D74和E202可增强热稳定性或压力稳定性。E450A突变和六重突变导致酶对压力不稳定。突变体的热失活数据表明，所有突变体对温度均有稳定作用。总之，突变体的压力稳定性和热稳定性证明，残基E202是HuAChE结构和功能稳定性的决定因素。
