Identification of two glutamic acid residues essential for catalysis in the β-glycosidase from the thermoacidophilic archaeon Sulfolobus solfataricus

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Abstract
The Sulfolobus solfataricus, strain MT4, β-glycosidase (Ssβgly) 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 Ssβ-gly domains. The Glu387 → Gin mutant showed no detectable activity, as expected for the residue acting as the nucleophile of the reaction. The Glu206 → Gin 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 plo-nitrophenyl-β-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.

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