FUT8 Catalysis Involves GDP-Fucose–Induced Loop Activation Promoting a Reaction at the S _N 1-S _N 2 Frontier

α1,6-Fucosyltransferase 8 (FUT8) catalyzes the core α1,6-fucosylation of <i>N</i>-glycans, a modification essential for the biological function of many mammalian glycoproteins. Despite its importance, the structural and mechanistic aspects of FUT8 catalysis remain incompletely understood. Here, we combine molecular dynamics, QM/MM, and metadynamics simulations to delineate the full catalytic cycle of FUT8. We reveal that GDP-fucose binding induces a concerted conformational rearrangement of two flexible loops, which cooperatively stabilize a closed, catalytically competent active site. Formation of the Michaelis complex primes the enzyme for fucose transfer via a slightly late and highly asynchronous S_N2 inverting mechanism. In fact, the reaction proceeds through a late transition state in a single kinetic step (energy barrier ∼18 kcal/mol, consistent with experimental <i>k</i> _cat values) but in three different stages, i.e.: (i) cleavage of the glycosidic bond between fucose and GDP, (ii) formation of the glycosidic bond and (iii) H-transfer from the acceptor to the catalytic Glu373, underscoring the asynchronous nature of this process. Moreover, topological calculations of the electron localization function (ELF) along the reaction coordinate reveal the transient formation of an intimate ion pair, with a lifetime of 350-800 fs, as a transient intermediate; notably, despite the cationic character of the transition state, no stable intermediate is formed. These findings highlight how structural rearrangements enable a chemically distinct catalytic process and provide a structural framework for rational inhibitor design.