Mechanism of human alpha-1,3-fucosyltransferase V : glycosidic cleavage occurs prior to nucleophilic attack

α-1,3-Fucosyltransferase V (FucT V) catalyzes the transfer of l-fucose from the donor sugar guanosine 5'-diphospho-β-l-fucose (GDP-Fuc) to an acceptor sugar. A secondary isotope effect on the fucosyltransfer reaction with guanosine 5'-diphospho-[1-²H]-β-l-fucose (GDP-[1-²H]-Fuc) as the substrate was observed and determined to be DV = 1.32 + 0.13 and DV/K = 1.27 + 0.07. Competitive inhibition of FucT V by guanosine 5'-diphospho-2-deoxy-2-fluoro-β-l-fucose (GDP-2F-Fuc) was observed with an inhibition constant of 4.2 íM which represents the most potent inhibitor of this enzyme to date. Incubation of GDP-2F-Fuc with FucT V and an acceptor molecule prior to the addition of GDP-Fuc had no effect on the potency of inhibition, indicating that GDP-2F-Fuc is neither an inactivator nor a slow substrate. Both the observed secondary isotope effect and the inhibition by GDP-2F-Fuc are consistent with a charged, sp²-hybridized, transition-state structure. A convenient and efficient synthesis of GDP-[1-²H]-Fuc and GDP-2F-Fuc and a nonradioactive, fluorescence assay for fucosyltransferase activity have been developed