Identification of two glutamic acid residues essential for catalysis in the beta-glycosidase from the thermoacidophilic archaeon Sulfolobus solfataricus.
In: Protein engineering, Jg. 9 (1996-12-01), Heft 12, S. 1191-5
academicJournal
Zugriff:
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.
Titel: |
Identification of two glutamic acid residues essential for catalysis in the beta-glycosidase from the thermoacidophilic archaeon Sulfolobus solfataricus.
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Autor/in / Beteiligte Person: | Moracci, M ; Capalbo, L ; Ciaramella, M ; Rossi, M |
Zeitschrift: | Protein engineering, Jg. 9 (1996-12-01), Heft 12, S. 1191-5 |
Veröffentlichung: | Oxford : Oxford University Press ; <i>Original Publication</i>: Oxford [Oxfordshire] ; Washington, DC : IRL Press, [c1986-c2003], 1996 |
Medientyp: | academicJournal |
ISSN: | 0269-2139 (print) |
DOI: | 10.1093/protein/9.12.1191 |
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