BARLEY BETA-D-GLUCAN EXOHYDROLASES WITH BETA-D-GLUCOSIDASE ACTIVITY -PURIFICATION, CHARACTERIZATION, AND DETERMINATION OF PRIMARY STRUCTURE FROM A CDNA CLONE

Two beta-glucan exohydrolases of apparent molecular masses 69,000 and 71,000 Da have been purified from extracts of 8-day germinated barley grains and are designated isoenzymes ExoI and ExoII, respectively. The sequences of their first 52 NH2-terminal amino acids show 64% positio nal identity. Both enzymes hydrolyze the (1,3)-beta-glucan, laminarin, but also hydrolyze (1,3; 1,4)-beta-glucan and 4-nitrophenyl beta-D-gl ucoside. The complete sequence of 602 amino acid residues of the matur e beta-glucan exohydrolase isoenzyme ExoII has been deduced by nucleot ide sequence analysis of a near full-length cDNA. Two other enzymes of apparent molecular mass 62,000 Da, designated beta I and beta II, wer e also purified from the extracts. Their amino acid sequences are simi lar to enzymes classified as beta-glucosidases and although they hydro lyze 4-nitrophenyl beta-glucoside, their substrate specificities and a ction patterns are more typical of polysaccharide exohydrolases of the (1,4)-beta-glucan glucohydrolase type. Both the beta-glucan exohydrol ase isoenzyme ExoI and the beta-glucosidase isoenzyme beta II release single glucosyl residues from the nonreducing ends of substrates and p roton-NMR shows that anomeric configurations are retained during hydro lysis by both classes of enzyme. These results raise general questions regarding the distinction between polysaccharide exohydrolases and gl ucosidases, together with more specific questions regarding the functi onal roles of the two classes of enzyme in germinating barley grain.