Henke Erik, Bornscheuer Uwe T, Schmid Rolf D, Pleiss Jürgen
Institute of Technical Biochemistry University of Stuttgart Allmandring 31 70569 Stuttgart, Germany.
Chembiochem. 2003 Jun 6;4(6):485-93. doi: 10.1002/cbic.200200518.
Carboxylesterases containing the sequence motif GGGX catalyze the hydrolysis of esters of chiral tertiary alcohols, albeit with only low to moderate enantioselectivity, for three model substrates (linalyl acetate, methyl-1-pentin-1-yl acetate, 2-phenyl-3-butin-2-yl acetate). In order to understand the molecular mechanism of enantiorecognition and to improve enantioselectivity for this interesting substrate class, the interaction of both enantiomers with the substrate binding sites of acetylcholinesterases and p-nitrobenzyl esterase from Bacillus subtilis was modeled and correlated to experimental enantioselectivity. For all substrate-enzyme pairs, enantiopreference and ranking by enantioselectivity could be predicted by the model. In p-nitrobenzyl esterase, one of the key residues in determining enantioselectivity was G105: exchange of this amino acid for an alanine residue led to a sixfold increase of enantioselectivity (E = 19) towards 2-phenyl-3-butin-2-yl acetate. However, the effect of this mutation is specific: the same mutant had the opposite enantiopreference towards the substrate linalyl acetate. Thus, depending on the substrate structure, the same mutant has either increased enantioselectivity or opposite enantiopreference compared to the wild-type enzyme.
含有序列基序GGG X的羧酸酯酶催化手性叔醇酯的水解反应，尽管对三种模型底物（乙酸芳樟酯、乙酸1-戊烯-1-基甲酯、乙酸2-苯基-3-丁炔-2-基酯）的对映选择性仅为低到中等。为了理解对映体识别的分子机制并提高对这类有趣底物的对映选择性，对两种对映体与枯草芽孢杆菌乙酰胆碱酯酶和对硝基苄酯酶的底物结合位点之间的相互作用进行了建模，并将其与实验对映选择性相关联。对于所有底物-酶对，该模型可以预测对映体偏好和对映选择性排序。在对硝基苄酯酶中，决定对映选择性的关键残基之一是G105：将该氨基酸替换为丙氨酸残基会使对乙酸2-苯基-3-丁炔-2-基酯的对映选择性提高六倍（E = 19）。然而，这种突变的影响具有特异性：相同的突变体对底物乙酸芳樟酯具有相反的对映体偏好。因此，根据底物结构，与野生型酶相比，相同的突变体要么对映选择性增加，要么对映体偏好相反。
