Contribution of subsites to catalysis and specificity in the extended binding cleft of Bacillus 1,3-1,4-β-D-glucan 4-glucanohydrolases

Specific low molecular weight oligosaccharides having a chromophoric aglycon were synthesized for kinetic evaluation of the enzyme catalysis. A subsite mapping study showed that the active site cleft is composed of four subsites on the non-reducing end, and allowed to estimate the contribution of subsites-II—-IV to transition state stabilization. In addition to a proper orientation of the scissile glycosidic bond of the substrate, inhibition kinetics with β-glucan- and cello-oligosaccharides suggest a binding preference of subsite -I for a 3-O-substituted over a 4-O-substituted glucopyranose unit. Subsite +I, on the other hand, has a minor contribution to binding but the nature of the aglycon in the substrate largely affects catalysis.