By means of QM(DFT)/MM metadynamics we have unraveled the hydrolytic reaction mechanism of amylosucrase (AS), a member of GH13 family. Our results provide an atomistic picture of the active site reorganization along the catalytic double-displacement reaction, clarifying whether the glycosyl-enzyme reaction intermediate features an α-glucosyl unit in an undistorted conformation, as inferred from structural studies, or a distorted -like conformation, as expected from mechanistic analysis of glycoside hydrolases (GHs). We show that, even though the first step of the reaction (glycosylation) results in a conformation, the α-glucosyl unit undergoes an easy conformational change toward a distorted conformation as the active site preorganizes for the forthcoming reaction step (deglycosylation), in which an acceptor molecule, i.e., a water molecule for the hydrolytic reaction, performs a nucleophilic attack on the anomeric carbon. The two conformations ( ad ) can be viewed as two different states of the glycosyl-enzyme intermediate (GEI), but only the state is preactivated for catalysis. These results are consistent with the general conformational itinerary observed for α-glucosidases.