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
M. Moracci et al., 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, BIOCHEM, 37(49), 1998, pp. 17262-17270
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.