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

The Sulfolobus solfataricus, strain MT4, ß-glycosidase(Ssßgly) is a thermophilic member of glycohydrolasefamily 1. To identify active-site residues, glutamic acids 206and 387 have been changed to isosteric glutamine by site-directedmutagenesis. Mutant proteins have been purified to homogeneityusing the Schistosoma japonicum glutathione S-transferase (GST)fusion system. The proteolytic cleavage of the chimeric proteinwith thrombin was only obtainable after the introduction ofa molecular spacer between the GST and the Ssß-glydomains. The Glu387 Gin mutant showed no detectable activity,as expected for the residue acting as the nucleophile of thereaction. The Glu206 Gin mutant showed 10- and 60-fold reducedactivities on aryl-galacto and aryl-glucosides, respectively,when compared with the wild type. Moreover, a significant K_mdecrease with plo-nitrophenyl-ß-D-glucoside was observed.The residual activity of the Glu206 Gln mutant lost the typicalpH dependence shown by the wild type. These data suggest thatGlu206 acts as the general acid/base catalyst in the hydrolysisreaction.