Mechanism of human alpha-1,3-fucosyltransferase V: glycosidic cleavage occurs prior to nucleophilic attack.
alpha-1,3-Fucosyltransferase V (FucT V) catalyzes the transfer of 1-fucose from the donor sugar guanosine 5'-diphospho-beta-1-fucose (GDP-Fuc) to an acceptor sugar. A secondary isotope effect on the fucosyltransfer reaction with guanosine 5'-diphospho-[1-2H]-beta-1-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-beta-1-fucose (GDP-2F-Fuc) was observed with an inhibition constant of 4.2 microM 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.
Murray
B W
BW
Department of Chemistry, Scripps Research Institute, La Jolla, California 92037, USA.
Wittmann
V
V
Burkart
M D
MD
Hung
S C
SC
Wong
C H
CH
D001665:Binding Sites; D005643:Fucose / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D005647:Fucosyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D015394:Molecular Structure; D013379:Substrate Specificity
D006153:Guanosine Diphosphate; D005643:Fucose; D005647:Fucosyltransferases; C031692:galactoside 3-fucosyltransferase