Mechanism and specificity of human alpha-1,3-fucosyltransferase V.
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.
Murray
B W
BW
Department of Chemistry, Scripps Research Institute, La Jolla, California 92037, USA.
Takayama
S
S
Schultz
J
J
Wong
C H
CH
D017485:1-Deoxynojirimycin / Q000031:analogs & derivatives; D000606:Amino Sugars / Q000378:metabolism; D002240:Carbohydrate Sequence; D002413:Cations, Divalent / Q000378:metabolism / Q000494:pharmacology; D017666:Deuterium Oxide / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005643:Fucose / Q000378:metabolism*; D005647:Fucosyltransferases / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006154:Guanosine Diphosphate Fucose / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D050112:Imino Pyranoses; D007700:Kinetics; D016256:Lewis X Antigen / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000494:pharmacology; D009844:Oligosaccharides / Q000138:chemical synthesis / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013402:Sugar Alcohols / Q000494:pharmacology
D000606:Amino Sugars; D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D050112:Imino Pyranoses; D016256:Lewis X Antigen; D009711:Nucleotides; D009844:Oligosaccharides; D011994:Recombinant Proteins; D013402:Sugar Alcohols; D006153:Guanosine Diphosphate; D006154:Guanosine Diphosphate Fucose; D017485:1-Deoxynojirimycin; D005643:Fucose; C000458:N-acetyllactosamine; C062662:deoxyfuconojirimycin; D005647:Fucosyltransferases; C031692:galactoside 3-fucosyltransferase; D017666:Deuterium Oxide