枯草芽孢杆菌内切葡聚糖酶Ala263位点突变对其活性的影响

Effect of Ala263 site mutagenesis on the activity of endoglucanase from Bacillus subtilis

研究通过对枯草芽孢杆菌内切葡聚糖酶celA进行三级结构模拟分析,发现其263位丙氨酸残基与底物纤维三糖间存在一定的作用;利用定点突变技术celA 263丙氨酸残基位点进行了不同的替换。结果显示野生酶的263位点丙氨酸(A263)突变为酪氨酸(A263Y)、异亮氨酸(A263I)、丝氨酸(A263S)和缬氨酸(A263V)后,其水解圈活性和CMC活力均大幅下降;DNS法测定的CMC活力分别由野生型的133.0U/L下降为A263Y 13.7U/L,A263I 36.1U/L,A263S 29.8U/L和A263V 19.6U/L。通过进一步的蛋白质精细结构模拟解析发现,263丙氨酸残基位点处于该蛋白活性口袋的内表面,为底物通道的结构区域,其羰基与底物糖基吡喃环上的羟基形成了分子间氢键。该氢键的形成可能对维持底物构象,正确引导底物在活性口袋中的定位起重要作用。该研究为诠释蛋白空间结构与其催化活性的内在联系奠定了一定的理论基础。

Structural models of endoglucanase （celA） from Bacillus subtilis revealed that Ala263 was positioned on the inner surface of protein and participated in the binding of sugars. Thus, the different variants with amino acid substitutions of A263Y, A263I, A263S and A263V were construct- ed by Site-directed mutagenesis. Using CMC as the substrate, all the mutant enzymes showed higher hydrolytic activities than the wild-type enzyme, which showed 133.0U/L of activity while A263Y, A263I, A263S and A263V showed 13.7U/L, 36.1U/L, 29.8U/L and 19.6U/L of activity, respective- ly. Further analysis revealed that A263 was located in the entrance of protein cleft and was involved in hydrogen bonding with glucopyranosyl rings of sugars, which could result in the formation of a larger active-site pocket and the right location of the substrate to increase catalytic activity. This study provided useful references for further research of the intrinsic relations between catalytic mechanism and its structure.