Mechanism and Specificity of Human α-1,3-Fucosyltransferase V

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Abstract
Human α-1,3-fucosyltransferase catalyzes the transfer of the l-fucose moiety from guanosine diphosphate−β-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 °C was accelerated from kobs = 3.5 × 10-6 to 3.8 × 10-5 min-1. Using the GDP−Fuc hydrolysis as the nonenzymatic rate, the enzymatic proficiency of FucT V, (kcat/Ki,GDP-fuc·Km,LacNAc)/knon, was estimated to be 1.2 × 1010 M-1 with a transition-state affinity of 8.6 × 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 Km,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 ≈ 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.