Lee Hee-Seob, Kim Jin-Soo, Shim Kyuho, Kim Jung-Woo, Inouye Kuniyo, Oneda Hiroshi, Kim Young-Wan, Cheong Kyung-Ah, Cha Hyunju, Woo Eui-Jeon, Auh Joong Hyuck, Lee Sung-Joon, Kim Jung-Wan, Park Kwan-Hwa
Center for Agricultural Biomaterials, and School of Agricultural Biotechnology, Seoul National University, Seoul, Korea.
FEBS J. 2006 Jan;273(1):109-21. doi: 10.1111/j.1742-4658.2005.05047.x.
As an effort to elucidate the quaternary structure of cyclomaltodextrinase I-5 (CDase I-5) as a function of pH and salt concentration, the dissociation/association processes of the enzyme were investigated under various pH and salt conditions. Previous crystallographic analysis of CDase I-5 indicated that it existed exclusively as a dodecamer at pH 7.0, forming an assembly of six 3D domain-swapped dimeric subunits. In the present study, analytical ultracentrifugation analysis suggested that CDase I-5 was present as a dimer in the pH range of 5.0-6.0, while the dodecameric form was predominant at pH values above 6.5. No dissociation of the dodecamer was observed at pH 7.0 and the above. Gel filtration chromatography showed that CDase I-5 dissociated into dimers at a rate of 8.58 x 10(-2) h(-1) at pH 6.0. A mutant enzyme with three histidine residues (H49, H89, and H539) substituted with valines dissociated into dimers faster than the wild-type enzyme at both pH 6.0 and 7.0. The tertiary structure indicated that the effect of pH on dissociation of the oligomer was mainly due to the protonation of H539. Unlike the pH-dependent process, the dissociation of wild-type CDase I-5 proceeded very fast at pH 7.0 in the presence of 0.2-1.0 M of KCl. Stopped-flow spectrophotometric analysis at various concentrations of KCl showed that the rate constants of dissociation (kd) from dodecamers into dimers were 5.96 s(-1) and 7.99 s(-1) in the presence of 0.2 M and 1.0 M of KCl, respectively.
为阐明环麦芽糊精酶I-5（CDase I-5）的四级结构与pH值和盐浓度的关系，研究了该酶在不同pH值和盐条件下的解离/缔合过程。先前对CDase I-5的晶体学分析表明，在pH 7.0时它仅以十二聚体形式存在，由六个三维结构域交换二聚体亚基组成一个聚集体。在本研究中，分析超速离心分析表明，CDase I-5在pH 5.0 - 6.0范围内以二聚体形式存在，而在pH值高于6.5时，十二聚体形式占主导。在pH 7.0及以上未观察到十二聚体的解离。凝胶过滤色谱显示，在pH 6.0时，CDase I-5以8.58×10⁻² h⁻¹的速率解离成二聚体。一种将三个组氨酸残基（H49、H89和H539）替换为缬氨酸的突变酶，在pH 6.0和7.0时解离成二聚体的速度都比野生型酶快。三级结构表明，pH对寡聚体解离的影响主要是由于H539的质子化。与pH依赖性过程不同，在0.2 - 1.0 M KCl存在下，野生型CDase I-5在pH 7.0时的解离非常快。在不同KCl浓度下的停流分光光度分析表明，在0.2 M和1.0 M KCl存在下，从十二聚体解离成二聚体的解离速率常数（kd）分别为5.96 s⁻¹和7.99 s⁻¹。
