Restoration of the activity of active-site mutants of the hyperthermophilic beta-glycosidase from Sulfolobus solfataricus: Dependence of the mechanism on the action of external nucleophiles

The beta-glycosidase from the hyperthermophilic Archaeon Sulfolobus solfata ricus hydrolyzes beta-glycosides following a retaining mechanism based upon the action of two amino acids: Glu387, which acts as the nucleophile of th e reaction, and Glu206, which acts as the general acid/base catalyst. The a ctivities of inactive mutants of the catalytic nucleophile Glu387Ala/Gly we re restored by externally added nucleophiles, Sodium azide and sodium forma te were used as external nucleophiles and the products of their reaction we re 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 Glu 387Gly mutant proceeded differently depending on the type of externally add ed nucleophile. Sodium azide restored the catalytic activity of the mutant by attacking the alpha-side of the anomeric carbon of the substrates, there by 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 biomim icking agent in replacing the natural nucleophile Glu387 is also discussed.