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

点击翻译按钮获取中文摘要

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.