Moracci M, Trincone A, Perugino G, Ciaramella M, Rossi M
Institute of Protein Biochemistry and Enzymology, Consiglio Nazionale delle Ricerche, Naples, Italy.
Biochemistry. 1998 Dec 8;37(49):17262-70. doi: 10.1021/bi981855f.
The beta-glycosidase from the hyperthermophilic Archaeon Sulfolobus solfataricus hydrolyzes beta-glycosides following a retaining mechanism based upon the action of two amino acids: Glu387, which acts as the nucleophile of the reaction, and Glu206, which acts as the general acid/base catalyst. The activities of inactive mutants of the catalytic nucleophile Glu387Ala/Gly were restored by externally added nucleophiles. Sodium azide and sodium formate were used as external nucleophiles and the products of their reaction were characterized. Glu387Ala/Gly mutants were reactivated with 2, 4-DNP-beta-Glc substrate and the Glu387Gly mutant showed recovered activity, with the same nucleophiles, also on 2-NP-beta-Glc. The reaction catalyzed by the Glu387Gly mutant proceeded differently depending on the type of externally added nucleophile. Sodium azide restored the catalytic activity of the mutant by attacking the alpha-side of the anomeric carbon of the substrates, thereby yielding an inverting glycosidase. Sodium formate promoted the opposite behavior (retaining) in the mutant, producing 3-O-beta-linked disaccharide derivative of the substrates. A possible role of sodium formate as a biomimicking agent in replacing the natural nucleophile Glu387 is also discussed.
嗜热古菌嗜热栖热菌（Sulfolobus solfataricus）中的β-糖苷酶通过基于两个氨基酸作用的保留机制水解β-糖苷：Glu387作为反应的亲核试剂，Glu206作为一般酸碱催化剂。催化亲核试剂Glu387Ala/Gly的无活性突变体的活性通过外部添加的亲核试剂得以恢复。叠氮化钠和甲酸钠用作外部亲核试剂，并对它们的反应产物进行了表征。用2,4-二硝基苯基-β-葡萄糖底物使Glu387Ala/Gly突变体重激活，并且Glu387Gly突变体在相同亲核试剂作用下对2-硝基苯基-β-葡萄糖也显示出恢复的活性。Glu387Gly突变体催化的反应根据外部添加亲核试剂的类型而有所不同。叠氮化钠通过攻击底物异头碳的α侧恢复了突变体的催化活性，从而产生一种转化糖苷酶。甲酸钠在突变体中促进了相反的行为（保留），产生底物的3-O-β-连接二糖衍生物。还讨论了甲酸钠作为生物模拟剂替代天然亲核试剂Glu387的可能作用。
