Enzymatic thioglycosylation: current knowledge and challenges - Carbohydrate Chemistry: Chemical and Biological Approaches - page 183

Actually more than 130 families have been identified w giving information
on mechanisms and protein folding. This CAZy classification is a
powerful tool to assign a family to a newly identified GH and determine
its putative catalytic residues.
2.1.1 Glycosidase mechanisms. In the past decades, GHs have been
extensively studied leading to their better understanding; especially two
main mechanisms have been characterized through X-ray crystallo-
graphic experiments. Hydrolysis of glycosidic bonds can be achieved with
inversion or retention of the anomeric configuration. 42
For inverting glycosidases, hydrolysis occurs following a one-step
displacement mechanism generated by two active residues (acting like
general acid/base) and a nucleophilic water molecule. In the active pocket
the two key carboxylic acids are approximately separated 10 Å from each
other enabling a nucleophilic attack on the opposite site of the glycosidic
linkage. 43 During the catalytic event, one of the key residues (general
base) activates the nucleophile water molecule, while the second carb-
oxylic acid (general acid) promotes the glycosidic leaving group departure
through acid catalysis (Fig. 5).
The second catalytic mechanism encountered in GHs is retaining
mechanism, occurring in a two-step double-displacement and resulting
in a double-inversion of the anomeric configuration. Both inverting and
retaining mechanisms occur via an oxycarbenium ion-like transition
state and involve two catalytic carboxylic acids, however retaining glyco-
sidases differ at some points: key residues are around 5.5 Å apart from
each other. 43 There is not enough space to allow simultaneous action of
incoming nucleophile molecule water and the carboxylates, as occurring
in inverting glycosidase mechanism.
The double-displacement involves the formation of a covalent glycosyl-
enzyme intermediate. The mechanism implicates first the acid-catalyzed
displacement of the leaving group (one of the key carboxylic acid acting
as a general acid) while the nucleophile residue (carboxylate) attacks the
anomeric center affording the covalent glycosyl-enzyme intermediate.
Then an incoming water molecule is activated by the general acid/base
Fig. 5 Inverting b -glycosidase mechanism.