SUBSITE AFFINITIES AND DISPOSITION OF CATALYTIC AMINO-ACIDS IN THE SUBSTRATE-BINDING REGION OF BARLEY 1,3-BETA-GLUCANASES - IMPLICATIONS INPLANT-PATHOGEN INTERACTIONS

Oligo-1,3-beta-glucosides with degrees of polymerization of 2-9 were l abeled at their reducing terminal residues by catalytic tritiation. Th ese substrates were used in detailed kinetic and thermodynamic analyse s to examine substrate binding in 1,3-beta-D-glucan glucanohydrolase ( EC 3.2,1.39) isoenzymes GI, GII, and GIII from young seedlings of barl ey (Hordeum vulgare). Bond-cleavage frequencies, together with the kin etic parameter k(cat)/K-m, have been calculated as a function of subst rate chain length to define the number of subsites that accommodate in dividual beta-glucosyl residues and to estimate binding energies at ea ch subsite, Each isoenzyme has eight beta-glucosyl-binding subsites. T he catalytic amino acids are located between the third and fourth subs ite from the nonreducing terminus of the substrate. Negative binding e nergies in subsites adjacent to the hydrolyzed glycosidic linkage sugg est that some substrate distortion may occur in this region during bin ding and that the resultant strain induced in the substrate might faci litate hydrolytic cleavage. If the 1,3-beta-glucanases exert their fun ction as pathogenesis-related proteins by hydrolyzing the branched or substituted 1,3; 1,6-beta-glucans of fungal walls, it is clear that re latively extended regions of the cell wall polysaccharide must fit int o the substrate-binding cleft of the enzyme.