Substrate binding and catalytic mechanism of a barley beta-D-Glucosidase/(1,4)-beta-D-glucan exohydrolase.
A beta-glucosidase, designated isoenzyme betaII, from germinated barley (Hordeum vulgare L.) hydrolyzes aryl-beta-glucosides and shares a high level of amino acid sequence similarity with beta-glucosidases of diverse origin. It releases glucose from the non-reducing termini of cellodextrins with catalytic efficiency factors, kcat/Km, that increase approximately 9-fold as the degree of polymerization of these substrates increases from 2 to 6. Thus, the enzyme has a specificity and action pattern characteristic of both beta-glucosidases (EC 3.2.1.21) and the polysaccharide exohydrolase, (1,4)-beta-glucan glucohydrolase (EC 3.2.1.74). At high concentrations (100 mM) of 4-nitrophenyl beta-glucoside, beta-glucosidase isoenzyme betaII catalyzes glycosyl transfer reactions, which generate 4-nitrophenyl-beta-laminaribioside, -cellobioside, and -gentiobioside. Subsite mapping with cellooligosaccharides indicates that the barley beta-glucosidase isoenzyme betaII has six substrate-binding subsites, each of which binds an individual beta-glucosyl residue. Amino acid residues Glu181 and Glu391 are identified as the probable catalytic acid and catalytic nucleophile, respectively. The enzyme is a family 1 glycoside hydrolase that is likely to adopt a (beta/alpha)8 barrel fold and in which the catalytic amino acid residues appear to be located at the bottom of a funnel-shaped pocket in the enzyme.
Hrmova
M
M
Department of Plant Science, University of Adelaide, Waite Campus, Glen Osmond, South Australia 5064, Australia.
MacGregor
E A
EA
Biely
P
P
Stewart
R J
RJ
Fincher
G B
GB
D000595:Amino Acid Sequence; D002384:Catalysis; D018360:Crystallography, X-Ray; D044643:Glucan 1,4-beta-Glucosidase; D001467:Hordeum / Q000201:enzymology*; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000737:chemistry / Q000378:metabolism*
D001617:beta-Glucosidase; D044643:Glucan 1,4-beta-Glucosidase