A tetrad of ionizable amino acids is important for catalysis in barley β-glucanases

Chen, Lin, Garrett, Thomas P. J., Fincher, Geoffrey B. and Hoj, Peter B. (1995) A tetrad of ionizable amino acids is important for catalysis in barley β-glucanases. Journal of Biological Chemistry, 270 14: 8093-8101. doi:10.1074/jbc.270.14.8093

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Author Chen, Lin
Garrett, Thomas P. J.
Fincher, Geoffrey B.
Hoj, Peter B.
Title A tetrad of ionizable amino acids is important for catalysis in barley β-glucanases
Journal name Journal of Biological Chemistry   Check publisher's open access policy
ISSN 0021-9258
Publication date 1995-04
Sub-type Article (original research)
DOI 10.1074/jbc.270.14.8093
Open Access Status File (Publisher version)
Volume 270
Issue 14
Start page 8093
End page 8101
Total pages 9
Place of publication Bethesda, MD, United States
Publisher American Society for Biochemistry and Molecular Biology
Language eng
Formatted abstract
Determination of the crystal structures of a 1,3-β-D-glucanase (E.C. and a 1,3-1,4-β-D-glucanase (E.C. from barley (Hordeum vulgare) (Varghese, J. N, Garrett, T. P. J., Colman, P. M., Chen, L., Høj, P. B., and Fincher, G. B.(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2785-2789) showed the spatial positions of the catalytic residues in the substrate-binding clefts of the enzymes and also identified highly conserved neighboring amino acid residues. Site-directed mutagenesis of the 1,3-β-glucanase has now been used to investigate the importance of these residues. Substitution of glutamine for the catalytic nucleophile Glu231 (mutant E231Q) reduced the specific activity about 20,000-fold. In contrast, substitution of glutamine for the catalytic acid Glu288 (mutant E288Q) had less severe consequences, reducing kcat approximately 350-fold with little effect on Km. Substitution of two neighboring and strictly conserved active site-located residues Glu279 (mutant E279Q) and Lys282 (mutant K282M) led to 240- and 2500-fold reductions of kcat, respectively, with small increases in Km. Thus, a tetrad of ionizable amino acids is required for efficient catalysis in barley β-glucanases. The active site-directed inhibitor 2,3-epoxypropyl β-laminaribioside was soaked into native crystals. Crystallographic refinement revealed all four residues (Glu231, Glu279, Lys282, and Glu288) to be in contact with the bound inhibitor, and the orientation of bound substrate in the active site of the glucanase was deduced.
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Non-UQ

Document type: Journal Article
Sub-type: Article (original research)
Collection: Office of the Vice-Chancellor
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