A -glucosidase, designated isoenzyme II, from germinated barley (Hordeum vulgare L.) hydrolyzes aryl--glucosides and shares a high level of amino acid sequence similarity with -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 -glucosidases (EC 3.2.1.21) and the polysaccharide exohydrolase, (1,4)--glucan glucohydrolase (EC 3.2.1.74). At high concentrations (100 mM) of 4-nitrophenyl -glucoside, -glucosidase isoenzyme II catalyzes glycosyl transfer reactions, which generate 4-nitrophenyl--laminaribioside, -cellobioside, and -gentiobioside. Subsite mapping with cellooligosaccharides indicates that the barley -glucosidase isoenzyme II has six substrate-binding subsites, each of which binds an individual -glucosyl residue. Amino acid residues Glu 181 and Glu 391 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 (/␣) 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.Two -glucosidases of apparent molecular mass 62,000 have been purified from extracts of germinated barley grain (1-3) and can be classified in the family 1 group of glycosyl hydrolases and related enzymes (4). The two enzymes have been designated isoenzymes I and II, and have isoelectric points of 8.9 and 9.0, respectively. Amino acid sequence analyses reveal a single amino acid difference in the first 50 NH 2 -terminal amino acid residues, and the complete amino acid sequence of isoenzyme II has been deduced from the nucleotide sequence of a corresponding cDNA clone (2).The function of the -glucosidases in the germinated barley grain has not been defined unequivocally, but will clearly be related to their substrate specificities. It has been widely assumed that -glucosidases prefer substrates of the type G-O-X, where G indicates the glucosyl residue and X can either be another glycosyl residue, for which the linkage position is not crucial, or a non-glycosyl aglycone group. As a result of the capacity of many -glucosidases to hydrolyze glucosides with a range of glycosyl or non-glycosyl aglycone groups, non-physiological substrates such as 4-nitrophenyl -D-glucopyranoside (4-NPG) 1 have been synthesized to measure activity in convenient spectrophotometric assays; the barley enzymes have also been assayed in this way. It has further been assumed that the rate of hydrolysis of oligomeric substrates by -glucosidases will remain approximately constant or decrease with increasing degree of polymerization (DP) of the substrate (5). However, the barley -glucosidase isoenzyme II hydrolyzes (1,4)--oligoglucosides much more efficiently than it hydrolyzes the aryl--gluco...