Substrate binding and catalytic mechanism of a Barley β-D- glucosidase/(1,4)-β-D-glucan exohydrolase

Abstract

A β-glucosidase, designated isoenzyme βII, from germinated barley (Hordeum vulgare L.) hydrolyzes aryl-β-glucosides and shares a high level of amino acid sequence similarity with β-glucosidases of diverse origin. It releases glucose from the non-reducing termini of cellodextrins with catalytic efficiency factors, k(cat)/K(m), 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 β-glucosidases (EC 3.2.1.21) and the polysaccharide exohydrolase, (1,4)-β-glucan glucohydrolase (EC 3.2.1.74). At high concentrations (100 mM) of 4-nitrophenyl β-glucoside, β-glucosidase isoenzyme βII catalyzes glycosyl transfer reactions, which generate 4-nitrophenyl-β- laminaribioside, -cellobioside, and -gentiobioside. Subsite mapping with cellooligosaccharides indicates that the barley β-glucosidase isoenzyme βII has six substrate-binding subsites, each of which binds an individual β- 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 (β/α)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.