BARLEY BETA-D-GLUCAN EXOHYDROLASES - SUBSTRATE-SPECIFICITY AND KINETIC-PROPERTIES

Two beta-D-glucan exohydrolases purified from germinated barley (Horde um vulgare) and designated isoenzymes ExoI and ExoII release glucose d uring the hydrolysis of a range of polymeric beta-D-glucans, beta-link ed oligo-D-glucosides, and aryl beta-D-glucosides. Of the polysacchari de substrates examined the enzymes show a preference for (1 --> 3)-bet a-glucans, although (1 --> 3;1 --> 6)- and (1 --> 3;1 --> 4)-beta-D-gl ucans are also hydrolysed. Oligosaccharides containing (1 --> 2)-, (1 --> 3)-, (1 --> 4)- and (1 --> 6)-beta-linked glucosyl residues are hy drolysed by both enzymes, which therefore exhibit a relatively broad s pecificity with respect to linkage positions in their substrates. Duri ng the hydrolysis of laminarabiose at high substrate concentrations (5 -20 mM), transglycosylation reactions can be detected. Both isoenzymes have a pH optimum of 5.25 and bell-shaped pH-activity curves. Detaile d kinetic analyses using the (1 --> 3)-beta-glucan, laminaran from Lam inaria digitata, allow the calculation of apparent K-m values of 98 an d 120 mu M, catalytic rate constants (k(cat)) of 73 and 28 sec(-1), an d catalytic efficiency factors (k(cat)/K-m) of 7.4 X 10(5) and 2.3 X 1 0(5) sec(-1) M-1 for isoenzymes ExoI and ExoII, respectively. The kine tic analyses also show a positive cooperativity of binding of the enzy mes for the barley (1 --> 3;1 --> 4)-beta-D-glucan, which suggests the presence of an allosteric substrate-binding site. Because of importan t differences between these barley enzymes and previously described (1 --> 3)-beta-D-glucan exohydrolases (EC 3.2.1.58) from other sources, they can not be readily assigned to existing Enzyme Commission groups. However, amino acid sequence similarities suggest that the enzymes ar e members of the family 3 group of glycosyl hydrolases. (C) 1998 Elsev ier Science Ltd.