Barley b-D-glucan exohydrolases. Substrate specificity and kinetic properties

Abstract

Two β-D-glucan exohydrolases purified from germinated barley (Hordeum vulgare) and designated isoenzymes ExoI and ExoII release glucose during the hydrolysis of a range of polymeric β-D-glucans, β-linked oligo-D- glucosides, and aryl β-D-glucosides. Of the polysacchide substrates examined the enzymes show a preference for (1 → 3)-β-glucans, although (1 → 3;1 → 6)- and (1 → 3;1 → 4)-β-D-glucans are also hydrolysed. Oligosaccharides containing (1 → 2)-, (1 → 3)-, (1 → 4)- and (1 → 6)-β-linked glucosyl residues are hydrolysed by both enzymes, which therefore exhibit a relatively broad specificity with respect to linkage positions in their substrates. During 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. Detailed kinetic analyses using the (1 → 3)-β-glucan, laminaran from Laminaria digitata, allow the calculation of apparent K(m) values of 98 and 120 μM, catalytic rate constants (k(cat)) of 73 and 28 sec-1, and catalytic efficiency factors (k(cat)/K(m)) of 7.4 x 105 and 2.3 x 105 sec-1 M-1 for isoenzymes ExoI and ExoII, respectively. The kinetic analyses also show a positive cooperativity of binding of the enzymes for the barley (1 → 3; 1 → 4)-β-D-glucan, which suggests the presence of an allosteric substrate- binding site. Because of important differences between these barley enzymes and previously described (1 → 3)-β-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 are members of the family 3 group of glycosyl hydrolases.