Identification of two glutamic acid residues essential for catalysis in the beta-glycosidase from the thermoacidophilic archaeon Sulfolobus solfataricus.
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.
Moracci
M
M
Institute of Protein Biochemistry and Enzymology-CNR, Naples, Italy.
Capalbo
L
L
Ciaramella
M
M
Rossi
M
M
D000595:Amino Acid Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics; D005959:Glucosidases / Q000235:genetics / Q000378:metabolism*; D005960:Glucosides / Q000378:metabolism; D018698:Glutamic Acid / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism; D013379:Substrate Specificity; D016944:Sulfolobus / Q000201:enzymology* / Q000235:genetics
D005960:Glucosides; D011993:Recombinant Fusion Proteins; C025193:4-nitrophenyl beta-D-glucoside; D018698:Glutamic Acid; D005959:Glucosidases; C104851:Sulfolobus solfataricus beta-glycosidase