Wang Jinhu, Zhang Rui, Liu Rutao, Liu Yongjun
College of Chemistry Chemical Engineering and Material Science, Zaozhuang University, Zaozhuang, Shandong 277160, China; School of Environmental Science and Engineering, Shandong University, 27 Shanda South Road, Jinan 250100, China.
School of Environmental Science and Engineering, Shandong University, 27 Shanda South Road, Jinan 250100, China.
J Mol Graph Model. 2014 Nov;54:100-6. doi: 10.1016/j.jmgm.2014.10.003. Epub 2014 Oct 22.
In the present study, the quantum mechanical/molecular mechanical (QM/MM) method was used to investigate the rescue mechanism of an E386G mutant as well as the glycosylation mechanism of the wild rice β-D-glucosidase. E386G mutant experiences an asynchronous collaborative process to glycosylate the anionic formate with an energy barrier of 22.6 kcal/mol, while the energy barrier is 25.9 kcal/mol for the wild complex. The low energy barrier of the mutated complex suggests that anionic formate might be a good nucleophile to attack the anomeric carbon atom. Both energy barriers can be lowered when the leaving departure releases from the active site, suggesting that the product release, rather than chemistry, contributes to the rate limiting in BGlu1 mutants. Structure analyses also indicate that the external nucleophile has little steric hindrance with pocket residues and adjusts freely to proceed the rescue mechanism of the mutated complex. Our calculations provide a guide for the selectivity of exogenous nucleophiles in the future study of β-glucosidase.
在本研究中，采用量子力学/分子力学（QM/MM）方法研究了E386G突变体的拯救机制以及野生型水稻β-D-葡萄糖苷酶的糖基化机制。E386G突变体经历了一个异步协同过程，使阴离子甲酸酯糖基化，能垒为22.6千卡/摩尔，而野生型复合物的能垒为25.9千卡/摩尔。突变复合物的低能垒表明阴离子甲酸酯可能是攻击异头碳原子的良好亲核试剂。当离去基团从活性位点释放时，两个能垒都可以降低，这表明产物释放而非化学反应是BGlu1突变体限速的原因。结构分析还表明，外部亲核试剂与口袋残基之间几乎没有空间位阻，并且可以自由调整以进行突变复合物的拯救机制。我们的计算为未来β-葡萄糖苷酶研究中外源亲核试剂的选择性提供了指导。
