AUTOMATED DOCKING OF ALPHA-(1,4)-LINKED AND ALPHA-(1,6)-LINKED GLYCOSYL TRISACCHARIDES IN THE GLUCOAMYLASE ACTIVE-SITE

Low-energy conformers of five alpha-(1,4)- and alpha-(1,6)-linked gluc osyl trisaccharides were flexibly docked into the glucoamylase active site using AutoDock 2.2. To ensure that all significant conformational space was searched, the starting trisaccharide conformers for docking were all possible combinations of the corresponding disaccharide low- energy conformers. All docked trisaccharides occupied subsites -1 and +1 in very similar modes to those of corresponding nonreducing-end dis accharides. For linear substrates, full binding at subsite +2 occurred only when the substrate reducing end was alpha-(1,4)-linked, with hyd rogen-bonding with the hydroxymethyl group being the only polar intera ction there. Given the absence of other important interactions at this subsite, multiple substrate conformations are allowed. For the one do cked branched substrate, steric hindrance in the alpha-(1,6)-glycosidi c oxygen suggests that the active-site residues have to change positio n for hydrolysis to occur. Subsite +1 of the glucoamylase active site allows flexibility in binding but, at least in Aspergillus glucoamylas es, subsite +2 selectively binds substrates alpha-(1,4)-linked between subsites +1 and +2. Enzyme engineering to limit substrate flexibility at subsite +2 could improve glucoamylase industrial properties.