Molecular Mechanism and Molecular Evolution of Glycosidases

α-葡萄糖苷酶（EC 3.2.1.20）是一种从底物中释放α-葡萄糖的外切型糖苷酶，分为两类（家族I和家族II），它们具有不同的结构和底物识别能力。还有糖苷酶，其结构与每个家族酶同源，如家族I的α-淀粉酶和家族II的α-木糖苷酶。本项目的目的是研究两个α-葡萄糖苷酶家族的结构与功能的关系，并了解葡萄糖苷酶的分子机制。1)鉴定了两个家族酶的催化氨基酸残基。并对其功能进行了分析。2)识别底物的糖基和糖苷基部分的氨基酸被鉴定。两个α-葡萄糖苷酶基团对糖基中羟基的识别能力不同。3)制备了酶的晶体。4)观察到α-二级动力学同位素效应，表明在催化反应中形成了氧代碳正离子中间体。5)通过分子生物学分析，阐明了两个α-葡萄糖苷酶家族的功能和结构，这对揭示与家族酶同源的葡萄糖苷酶的分子机制具有重要意义。

α-Glucosidase (EC 3.2.1.20), an exo-type glycosidase releasing α-glucose from substrate, is divided into two types of groups (family I and family II) that have different structures and substrate recognition. There are also the glycosidases, of which structures are homologous to each of family enzyme, such as α-amylase for family I and α-xylosidase for family II. The purpose of the project is to investigate the relationship of structure and function of two α-glucosidase families, and to understand the molecular mechanism of glycosidases. 1)The catalytic amino acid residues of two family enzymes were identified. Their functions were also analyzed. 2)The amino acids recognizing the glycon and aglycon moieties of substrate were identified. Two α-glucosidase groups showed the different recognition against the hydroxyl group in the glycon. 3)The crystal of enzyme was prepared. 4)The α-secondary kinetic isotope effects were observed, suggesting the formation of oxo-carbenium ion intermediate in the catalytic reaction. 5)The molecular analysis succeeded in elucidating the function and structure of two α-glucosidase families, and the findings are important to reveal the molecular mechanism of glycosidases homologous to family enzymes.