The Sulfolobus solfataricus, strain MT4, beta-glycosidase (Ss beta-gly) is a thermophilic member of glycohydrolase family 1. To identify active-site residues, glutamic acids 206 and 387 have been changed to isosteric glutamine by site-directed mutagenesis. Mutant proteins have been purified to homogeneity using the Schistosoma japonicum glutathione S-transferase (GST) fusion system. The proteolytic cleavage of the chimeric protein with thrombin was only obtainable after the introduction of a molecular spacer between the GST and the Ss beta-gly domains. The Glu387-->Gln mutant showed no detectable activity, as expected for the residue acting as the nucleophile of the reaction. The Glu206-->Gln mutant showed 10- and 60-fold reduced activities on aryl-galacto and aryl-glucosides, respectively, when compared with the wild type. Moreover, a significant Km decrease with p/o-nitrophenyl-beta-D-glucoside was observed. The residual activity of the Glu206-->Gln mutant lost the typical pH dependence shown by the wild type. These data suggest that Glu206 acts as the general acid/base catalyst in the hydrolysis reaction.