A TETRAD OF IONIZABLE AMINO-ACIDS IS IMPORTANT FOR CATALYSIS IN BARLEY BETA-GLUCANASES

Determination of the crystal structures of a 1,3-beta-D-glucanase (E.C . 3.2.1.39) and a 1,3-1,4-beta-D-glucanase (E.C. 3.2.1.73) from barley (Hordeum vulgare) (Varghese, J. N, Garrett, T. P. J., Cohnan, P. M., Chen, L., Hoj, 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 iden tified highly conserved neighboring amino acid residues. Site-directed mutagenesis of the 1,3-beta-glucanase has now been used to investigat e the importance of these residues. Substitution of glutamine for the catalytic nucleophile Glu(231) (mutant E231Q) reduced the specific act ivity about 20,000-fold. In contrast, substitution of glutamine for th e catalytic acid Glu(288) (mutant E288Q) had less severe consequences, reducing k(cat) approximately 350-fold with little effect on K-m. Sub stitution of two neighboring and strictly conserved active site-locate d residues Glu(279) (mutant E279Q) and Lys(282) (mutant K282M) led to 240- and 2500-fold reductions of k(cat), respectively, with small incr eases in K-m. Thus, a tetrad of ionizable amino acids is required for efficient catalysis in barley beta-glucanases. The active site-directe d inhibitor 2,3-epoxypropyl beta-laminaribioside was soaked into nativ e crystals. Crystallographic refinement revealed all four residues (Gl u(231), Glu(279), Lys(282), and Glu(288)) to be in contact with the bo und inhibitor, and the orientation of bound substrate in the active si te of the glucanase was deduced.