Subsite affinities and disposition of catalytic amino acids in the substrate-binding region of barley 1,3-beta-glucanases. Implications in plant-pathogen interactions.
Oligo-1,3-beta-glucosides with degrees of polymerization of 2-9 were labeled at their reducing terminal residues by catalytic tritiation. These substrates were used in detailed kinetic and thermodynamic analyses 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 barley (Hordeum vulgare). Bond-cleavage frequencies, together with the kinetic parameter kcat/Km, have been calculated as a function of substrate chain length to define the number of subsites that accommodate individual beta-glucosyl residues and to estimate binding energies at each subsite. Each isoenzyme has eight beta-glucosyl-binding subsites. The catalytic amino acids are located between the third and fourth subsite from the nonreducing terminus of the substrate. Negative binding energies in subsites adjacent to the hydrolyzed glycosidic linkage suggest that some substrate distortion may occur in this region during binding and that the resultant strain induced in the substrate might facilitate hydrolytic cleavage. If the 1,3-beta-glucanases exert their function as pathogenesis-related proteins by hydrolyzing the branched or substituted 1,3;1,6-beta-glucans of fungal walls, it is clear that relatively extended regions of the cell wall polysaccharide must fit into the substrate-binding cleft of the enzyme.
Hrmova
M
M
Department of Plant Science, University of Adelaide, Australia.
Garrett
T P
TP
Fincher
G B
GB
D000596:Amino Acids / Q000378:metabolism*; D002384:Catalysis; D043326:Glucan 1,3-beta-Glucosidase; D001467:Hordeum / Q000201:enzymology* / Q000382:microbiology; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D013379:Substrate Specificity; D013816:Thermodynamics; D001617:beta-Glucosidase / Q000737:chemistry / Q000378:metabolism*
D000596:Amino Acids; D001617:beta-Glucosidase; D043326:Glucan 1,3-beta-Glucosidase