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克氏锥虫唾液酸转移酶(TcTs)是恰加斯病的致病原,它具有由6个β片构成的桶状催化结构域。该催化结构域集中在酶N端的边缘。本文利用量子力学/分子力学(QM/MM)联用的模型研究了克氏锥虫唾液酸转移酶的催化机理。初始酶和底物复合物模型由蛋白质晶体数据库得到(PDB ID:lSOI)。其中QM部分在半经验模型中由AM1描述,在从头算模型中由B3LYP/6-3lG~*描述。MM部分只取酶的N端结构域,并始终由AMBER力场来描述。QM部分与MM部分成键相互作用边界用pseudo-bond方法处理,将3个重要的氨基酸残基(Glu230,Asp59,Tyr342)的C_β-C_β键作为OM/MM模型中的pseudo-bond。由Nudged Elastic Band(NEB)路径优化方法得到的TcTs半经验的最低能量反应路径中,关键原子间距离沿最低能量路径的变化表明:反应开始后Glu230开始靠近Tyr342,当它们之间的氢键距离由2.9 A缩短为2.4 A时,Tyr342将质子转移给Glu230,增强了Tyr342酚氧负离子的碱性,更有利于Tyr342亲核进攻糖苷键。同时,Asp59作为酸,提供质子给糖苷键断裂后的离去基团。过程中,伴随着唾液酸的单糖糖环从扭曲的船式构象向松弛的椅式构象的转变,从而更有利于稳定生成的共价唾液酸-酶中间产物。对得到的半经验的最低能量反应路径再做B3LYP/6-31G~*/MM模型下的优化,得到反应的能垒约为13.53 kcal/mol,说明该反应路径是合理的。研究结果与实验上通过突变的TcTs_(D59A)推测的乒乓双置换酸碱催化的机理一致,是对实验结论的有力支持,为TcTs抑制剂的设计和结构修饰提供了理论参考,有助于预防和抗恰加斯病的新药物研发。
Trypanosoma sialis transferase (TcTs), the causative agent of Chagas disease, has a barrel-shaped catalytic domain made up of six beta sheets. This catalytic domain is concentrated at the N-terminal edge of the enzyme. In this paper, the catalytic mechanism of Trypanosoma cruzi sialyltransferase was studied by using the model of quantum mechanics / molecular mechanics (QM / MM). The initial enzyme and substrate complex model was obtained from the Protein Crystal Database (PDB ID: lSOI). The QM part is described by AM1 in semi-empirical model and B3LYP / 6-3lG ~ * in ab initio model. The MM moiety only takes the N-terminal domain of the enzyme and is always described by the AMBER force field. The interaction between the QM part and the MM part was treated by the pseudo-bond method. The C_β-C_β bonds of three important amino acid residues (Glu230, Asp59, Tyr342) were regarded as pseudo-bonds in the OM / MM model. In the semiempirical least-energy reaction path of TcTs derived from the Nudged Elastic Band (NEB) path optimization method, the change of the distance between the key atoms along the lowest energy path shows that Glu230 starts to approach Tyr342 after the reaction starts, and when the hydrogen bond distance When 2.9 A was shortened to 2.4 A, Tyr342 transferred protons to Glu230, which enhanced the basicity of Tyr342 phenoxy anion and facilitated nucleophilic attack on the glycosidic bond of Tyr342. In the meantime, Asp59 acts as an acid, providing a proton to the leaving group after cleavage of the glycosidic bond. Accompanied by a shift of the monosaccharide sugar ring of sialic acid from a twisted, helical conformation to a relaxed, chair conformation, favoring the stable formation of covalent sialic acid-enzyme intermediates. Under the B3LYP / 6-31G ~ * / MM model, the obtained semi-empirical minimum energy reaction path is optimized and the energy barrier of the reaction is about 13.53 kcal / mol, which shows that the reaction path is reasonable. The results are consistent with the experimentally determined mechanism of acid-base catalysis of pTAP by the mutated TcTs_ (D59A), which is a strong support for the experimental conclusion and provide a theoretical reference for the design and structural modification of TcTs inhibitors, which is helpful to prevent And anti-Chagas disease drug development.