Yuan Y R, Xia Z X, Yang C H, Yang B H, Yeh M
Shanghai Institute of Cell Biology, Chinese Academy of Sciences, China.
Cell Res. 1998 Sep;8(3):219-30. doi: 10.1038/cr.1998.22.
The mechanism of ester hydrolysis has been extensively studied; however, the precise function of active-site residues in promoting catalysis is unclear. We describe here the structural models for the complex of a catalytic antibody Fv fragment with a phosphonate transition-state analogue, constructed by using gene cloning, sequencing and molecular modeling, mainly based on a known X-ray structure of a catalytic antibody. Hydrophobic and electrostatic analyses of the Fv/analog and Fv/substrate interaction suggest the hydrolysis mechanism: Tyr L91 and Tyr H97 play important roles to stabilize the beta-naphthyl group of hapten through pi-stack; His H35 donates a pair of free electrons at the atom NE2 to an active water and let it to be a partial hydroxide, which attacks the carbon atom of the carbonyl group of the substrate. Both His H35 and Arg L96 can form hydrogen bonds and stabilize the anionic tetrahedral intermediate formed during turnover. This mechanism emphasizes that an active water bridge may be formed during hydrolysis process.
酯水解的机制已得到广泛研究；然而，活性位点残基在促进催化中的精确功能尚不清楚。我们在此描述了一种催化抗体Fv片段与膦酸酯过渡态类似物复合物的结构模型，该模型通过基因克隆、测序和分子建模构建，主要基于催化抗体的已知X射线结构。对Fv/类似物和Fv/底物相互作用的疏水和静电分析表明了水解机制：Tyr L91和Tyr H97通过π-堆积在稳定半抗原的β-萘基方面发挥重要作用；His H35在原子NE2处向活性水提供一对自由电子，使其成为部分氢氧化物，后者攻击底物羰基的碳原子。His H35和Arg L96都可以形成氢键并稳定周转过程中形成的阴离子四面体中间体。该机制强调在水解过程中可能形成活性水桥。
