Human alpha-1,3-fucosyltransferase catalyzes the transfer of the L-fucose moiety from guanosine diphosphate-beta-L-fucose (GDP-Fuc) to acceptor sugars to form biologically important fucoglycoconjugates, including sialyl Lewis x (SLex). Evidence for a general base mechanism is supported by a pH-rate profile that revealed a catalytic residue with a pKa of 4.1. The characterized solvent kinetic isotope effect (Dv = 2.9, Dv/k = 2.1) in a proton inventory study indicates that only one-proton transfer is involved in the catalytic step leading to the formation of the transition state. Evidence for Mn2+ as an electrophilic catalyst was supported by the observation that the nonenzymatic transfer of L-fucose from GDP-Fuc to the hydroxyl group of water in the presence of 10 mM MnCl2 at 20 degrees C was accelerated from K(obs)= 3.5 x 10(-6) to 3.8 x 10(-5) min-1. Using the GDP-Fuc hydrolysis as the nonenzymatic rate, the enzymatic proficiency of FucT V, (Kcat/Ki,GDP-fuc. K(m),1.acNAc)/K(non), was estimated to be 1.2 x 10(10) M-1 with a transition-state affinity of 8.6 x 10(-11) M. The Km for Mn2+ was determined to be 6.1 mM, and alternative divalent metal cofactors were identified as Ca2+, Co2+, and Mg2+. Detailed kinetic characterization of the acceptor sugar specificity indicated that incorporation of hydrophobic functionality [e.g. -O-(CH2)5CO2CH3] to the reducing end of the acceptor sugar substantially decreased the K(m),acceptor by over 100-fold. The role of the nucleotide was investigated by studying the inhibition of nucleotides, including the guanosine series. The inhibitory potency trend (GTP approximately GDP > GMP > > guanosine) is consistent with bidentate chelation of Mn2+ by GDP-Fuc. The role of charge and distance in the synergistic inhibitory effect by the combination of GDP, an aza sugar, and the acceptor sugar was probed. A mechanism for fucosyl transfer incorporating these findings is proposed and discussed.