A GDP-fucose-protected, pyridoxal-5'-Phosphate/NaBH(4)-sensitive lys residue common to human alpha1-->3Fucosyltransferases corresponds to Lys(300) in FucT-IV.
Human alpha1-->3/4fucosyltransferases (FucTs) contain a common essential pyridoxal-5'-phosphate(PLP)/NaBH(4) reactive, GDP-fucose-protectable Lys. For identification, site-directed mutants at lysines of FucT-IV and -VII were prepared and tested. Non conserved lysine mutants K119Y and K394Q were similar to wild-type FucT-IV. However, mutants of conserved lysines K228R and K300R were distinct. The specific activity of K228R was 2- to 3-fold lower but retained K(m) values for donor and acceptor substrates as wild-type FucT-IV. The specific activity of K300R was reduced over 400-fold with an apparent K(m) for GDP-fucose over 200 microM. FucT-VII mutants K169R and K240R (equivalent to K228R and K300R for FucT-IV, respectively) were inactive. No change in PLP/NaBH(4) sensitivity occurred with K119Y, K228R, and K394Q compared to wild-type FucT-IV. These and previous results (A. L. Sherwood, A. T. Nguyen, J. M. Whitaker, B. A. Macher, M. R. Stroud, and E. H. Holmes, J. Biol. Chem. 273, 25256-25260, 1998) demonstrate that of three conserved lysines in FucT-IV, two (Lys(228) and Lys(283)) are not involved in substrate binding but perhaps in catalysis. The third site, Lys(300), is involved in GDP-fucose binding and PLP/NaBH(4) inactivation.
Sherwood
A L
AL
Department of Cell Surface Biochemistry, Molecular Medicine, Northwest Hospital, 2203 Airport Way South, Suite 200, Seattle, Washington 98134, USA.
Davis
W C
WC
Ho
S
S
Macher
B A
BA
Stroud
M R
MR
Upchurch
D A
DA
Holmes
E H
EH
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D001894:Borohydrides / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019556:COS Cells; D017931:DNA Primers; D005647:Fucosyltransferases / Q000737:chemistry / Q000378:metabolism*; D006154:Guanosine Diphosphate Fucose / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D011732:Pyridoxal Phosphate / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D001894:Borohydrides; D017931:DNA Primers; D006154:Guanosine Diphosphate Fucose; D011732:Pyridoxal Phosphate; C025364:sodium borohydride; D005647:Fucosyltransferases; D008239:Lysine