Wang Jinhu, Pengthaisong Salila, Cairns James R Ketudat, Liu Yongjun
Key Lab of Theoretical and Computational Chemistry in University of Shandong, School of Chemistry and Chemical Engineering, Shandong University, Jinan, Shandong 250100, China.
Biochim Biophys Acta. 2013 Feb;1834(2):536-45. doi: 10.1016/j.bbapap.2012.11.003. Epub 2012 Nov 19.
Nucleophile mutants of retaining β-glycosidase can act as glycosynthases to efficiently catalyze the synthesis of oligosaccharides. Previous studies proved that rice BGlu1 mutants E386G, E386S and E386A catalyze the oligosaccharide synthesis with different rates. The E386G mutant gave the fastest transglucosylation rate, which was approximately 3- and 19-fold faster than those of E386S and E386A. To account for the differences of their activities, in this paper, the X-ray crystal structures of BGlu1 mutants E386S and E386A were solved and compared with that of E386G mutant. However, they show quite similar active sites, which implies that their activities cannot be elucidated from the crystal structures alone. Therefore, a combined quantum mechanical/molecular mechanical (QM/MM) calculations were further performed. Our calculations reveal that the catalytic reaction follows a single-step mechanism, i.e., the extraction of proton by the acid/base, E176, and the formation of glycosidic bond are concerted. The energy barriers are calculated to be 19.9, 21.5 and 21.9kcal/mol for the mutants of E386G, E386S and E386A, respectively, which is consistent with the order of their experimental relative activities. But based on the calculated activation energies, 1.1kcal/mol energy difference may translate to nearly 100 fold rate difference. Although the rate limiting step in these mutants has not been established, considering the size of the product and the nature of the active site, it is likely that the product release, rather than chemistry, is rate limiting in these oligosaccharides synthesis catalyzed by BGlu1 mutants.
保留型β-糖苷酶的亲核突变体可作为糖基合成酶，有效催化寡糖的合成。先前的研究证明，水稻β-葡萄糖苷酶1（BGlu1）突变体E386G、E386S和E386A以不同速率催化寡糖合成。E386G突变体的转糖基化速率最快，比E386S和E386A分别快约3倍和19倍。为了解释它们活性的差异，本文解析了BGlu1突变体E386S和E386A的X射线晶体结构，并与E386G突变体的结构进行了比较。然而，它们的活性位点非常相似，这意味着仅从晶体结构无法阐明它们的活性。因此，进一步进行了量子力学/分子力学（QM/MM）联合计算。我们的计算表明，催化反应遵循单步机制，即酸碱E176提取质子和糖苷键的形成是协同进行的。计算得出E386G、E386S和E386A突变体的能垒分别为19.9、21.5和21.9kcal/mol，这与它们的实验相对活性顺序一致。但根据计算出的活化能，1.1kcal/mol的能量差异可能转化为近100倍的速率差异。尽管这些突变体中的限速步骤尚未确定，但考虑到产物的大小和活性位点的性质，在BGlu1突变体催化的这些寡糖合成中，产物释放而非化学反应可能是限速步骤。
