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., 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-beta-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 beta-glucanases. The active site-directed inhibitor 2,3-epoxypropyl beta-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.
Chen
L
L
Department of Biochemistry, La Trobe University, Bundoora, Victoria, Australia.
Garrett
T P
TP
Fincher
G B
GB
Høj
P B
PB
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D001483:Base Sequence; D002240:Carbohydrate Sequence; D002352:Carrier Proteins / Q000235:genetics; D002384:Catalysis; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D043326:Glucan 1,3-beta-Glucosidase; D001467:Hordeum / Q000201:enzymology*; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics; D001617:beta-Glucosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D000596:Amino Acids; D002352:Carrier Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D001617:beta-Glucosidase; D043326:Glucan 1,3-beta-Glucosidase