Analyzes for Mechanism of Substate Recognition and Structure of Sugar Chain in Fungus Crystalline alpha-Glucosidase

黑曲霉α-葡萄糖苷酶(ANGase)是一种“麦芽糖酶”型酶，由p1(227个氨基酸)和P2(719个氨基酸)两个不同的亚基组成，不通过S-S桥连接。通过Edman降解和遗传方法确定了它们的一级结构。基因组核苷酸序列显示，P1和P2多肽按顺序由单一阅读框编码。在两个亚基之间去除了14个氨基酸，这表明这是一种独特的翻译后修饰，蛋白质降解有限。P2的天冬氨酸(224)是一种催化氨基酸。分离得到7个N-连接的高甘露糖型低聚糖，并对其结构进行了鉴定。四条糖链含有一个通过α-(1*2)-键与Man(A)结合的呋喃半乳糖基。其中三种是新型的N-连接低聚糖。O-连接糖也是从ANGase中分离出来的。两个化合物的结构分析表明，甘露糖和α-甘露糖基-(1*2)-甘露糖直接与ANGase的丝氨酸/苏氨酸结合。在此基础上，比较了黑曲霉α-葡萄糖苷酶和其他来源的α-葡萄糖苷酶的一级结构。α-葡萄糖苷酶在一级结构上分为两个家族。在分析D-葡萄糖醛水合反应和α-葡萄糖苷酶和糖淀粉酶的α-次级动力学同位素效应的基础上，讨论了碳水化合物水解酶的底物识别模式和反应机理模型。

An crystalline alpha-glucosidase from Asperillus niger (ANGase), a so-called "maltase"-type enzyme, was composed of two different subunits, p1 (227 amino acids) and P2 (719 amino acids), which were not linked via S-S bridge. Their primary structures were determined by Edman degradation and genetic methods. The genomic nucleotide sequence revealed theat the P1 and P2 polypeptides were encoded by a single reading frame in this order. Between two subunits 14 amino acids were removed, suggesting an unique post-translational modification with limited proteolysis. The Asp (224) of P2 was identified to be one of catalytic amino acids. Seven N-linked high-mannose type oligosaccharides were isolated, and their structures were determined. Four sugar chains contained one galactofuranosyl residue bound via alpha- (1*2) -linkage to Man (A). Three of them are novel types of N-linked oligosaccharides. The O-linked sugars also were isolated from ANGase. Structural analysis of two compounds indicated that mannose and alpha-mannosyl- (1*2) -mannose bound directly to Ser/Thr of ANGase.Based on the above findings, A.niger alpha-glucosidase was compared in the primary structures with alpha-glucosidases from other origins. alpha-Glucosidases were elucidated to be classified into the two families on the primary structures. In adittion, the mode of substrate recognition and the model of reaction mechanism in the carbohydrate-hydrolases were discussed and proposed on the analysis for the hydration reaction of D-glucal and the alpha-secondary kinetic isotope effects by alpha-glucosidase and glucoamylase.