Another important reactant is the nucleophilic water molecule, which donates a hydroxyl ion to the anomeric carbon atom at subsite −1, and has been predicted to stabilize the oxocarbenium-type transition state of the central sugar ring at subsite −1 after glycosidic bond cleavage [24].
In the equilibrated system, the carboxylate oxygen atom of Glu87 forms a strong, stable hydrogen bond with the OH-4 atom of the cellobiose fragment at subsite +1.
A variety of putative reaction coordinates were examined, but it was found to be impossible to locate a stable reactant structure in which the nucleophilic water molecule is at a reasonable distance from the anomeric carbon at subsite −1 and also makes a strong hydrogen bonding interaction with the base catalyst [24].
Thus Glu636 of CtCBP recognized the C1 hydroxyl of β-glucose at subsite +1, while in CtCDP the presence of Ala800 conferred more space, which allowed accommodation of C1 substituted disaccharide acceptors at the corresponding subsites +1 and +2.
Its role might be to force the bend that occurs in the polysaccharide chain on binding, thus favoring substrate distortion at subsite −1.
Subsite map determined by kinetic analysis of rAaBGL1 for cellooligosaccharides hydrolysis, using the method for subsite analysis of exo-acting enzyme (Hiromi et al. 1973), revealed that AaBGL1 had the subsite +2 like other GH family 3 BGLs, but subsite map for laminarioligosaccharides, showed weak affinity at subsite +2 (Figure 6).
This residue may stack onto substrate at subsite +3 in BXA43, as supported by homology modelling (Figure 4).
Tyr351 was located about 5.3Å away from the C1 at subsite −1 and Glu87 and Asp255 made hydrogen bonding interactions with O3 at subsite +1 and O2 at subsite −1 of the carbohydrate, respectively.
In the crystal structure of liganded CelA [24], a Family 8 inverting enzyme with Glu95 as the proton donor and Asp278 as base catalyst, a potentially nucleophilic water molecule was seen in the electron density map, making a strong hydrogen bonding interaction with the base catalyst at a distance of 2.8 Å from C1 at subsite −1.
We examine also how the pyranose ring at the catalytic center (at subsite −1) may undergo conformational changes to facilitate the reaction.
