Glycoside hydrolases of families 32 (GH32) and 68 (GH68) belong to clan GH-J, containing hydrolytic enzymes (sucrose/
fructans as donor substrates) and fructosyltransferases (sucrose/fructans as donor and acceptor substrates). In GH32
members, some of the sugar substrates can also function as inhibitors, this regulatory aspect further adding to the
complexity in enzyme functionalities within this family. Although 3D structural information becomes increasingly available
within this clan and huge progress has been made on structure-function relationships, it is not clear why some sugars bind
as inhibitors without being catalyzed. Conserved aspartate and glutamate residues are well known to act as nucleophile and
acid/bases within this clan. Based on the available 3D structures of enzymes and enzyme-ligand complexes as well as
docking simulations, we calculated the pKa of the acid-base before and after substrate binding. The obtained results
strongly suggest that most GH-J members show an acid-base catalyst that is not sufficiently protonated before ligand
entrance, while the acid-base can be fully protonated when a substrate, but not an inhibitor, enters the catalytic pocket.
This provides a new mechanistic insight aiming at understanding the complex substrate and inhibitor specificities observed
within the GH-J clan. Moreover, besides the effect of substrate entrance on its own, we strongly suggest that a highly conserved arginine residue (in the RDP motif) rather than the previously proposed Tyr motif (not conserved) provides the proton to increase the pKa of the acid-base catalyst.