Starch and Cyclodextrin Research Unit, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand.
Starch and Cyclodextrin Research Unit, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand; Department of Chemistry, Faculty of Science, Ramkhamhaeng University, Bangkok 10240, Thailand.
Int J Biol Macromol. 2015 May;76:230-5. doi: 10.1016/j.ijbiomac.2015.02.035. Epub 2015 Mar 4.
This work aims to identify essential tryptophan residue(s) of amylomaltase from Corynebacterium glutamicum (CgAM) through chemical modification and site-directed mutagenesis techniques. The recombinant enzyme expressed by Escherichia coli was purified and treated with N-bromosuccinimide (NBS), a modifying agent for tryptophan. A significant decrease in enzyme activity was observed indicating that tryptophan is important for catalysis. Inactivation kinetics with NBS resulted in pseudo first-order rate constant (kinact) of 2.31 min(-1). Substrate protection experiment confirmed the active site localization of the NBS-modified tryptophan residue(s) in CgAM. Site-directed mutagenesis was performed on W330, W425 and W673 to localize essential tryptophan residues. Substitution by alanine resulted in the loss of intra- and intermolecular transglucosylation activities for all mutated CgAMs. Analysis of circular dichroism spectra showed no change in the secondary structure of W425A but a significant change for W330A and W673A from that of the WT. From these results in combination with X-ray structural data and interpretation from the binding interactions in the active site region, W425 was confirmed to be essential for catalytic activity of CgAM. The hydrophobicity of this tryptophan was thought to be critical for substrate binding and supporting catalytic action of the three carboxylate residues at the active site.
本工作旨在通过化学修饰和定点突变技术鉴定来自谷氨酸棒杆菌（Corynebacterium glutamicum）的支链淀粉酶（amylomaltase）的必需色氨酸残基（s）。通过大肠杆菌表达的重组酶被纯化，并使用 N-溴代丁二酰亚胺（NBS）处理，NBS 是一种色氨酸修饰剂。观察到酶活性显著下降，表明色氨酸对于催化很重要。用 NBS 进行的失活动力学导致假一级速率常数（kinact）为 2.31 min(-1)。底物保护实验证实了 NBS 修饰的色氨酸残基（s）在 CgAM 中的活性位点定位。对 W330、W425 和 W673 进行定点突变以定位必需的色氨酸残基。所有突变的 CgAMs 中，丙氨酸取代导致内-和分子间转葡糖苷活性丧失。圆二色性光谱分析表明，W425A 的二级结构没有变化，但 W330A 和 W673A 的二级结构与 WT 的相比有明显变化。从这些结果结合 X 射线结构数据和对活性位点区域结合相互作用的解释，证实 W425 对于 CgAM 的催化活性是必需的。该色氨酸的疏水性被认为对于底物结合和支持活性位点处的三个羧酸盐残基的催化作用至关重要。
