Human alpha 1-->3/4fucosyltransferases (FucTs) contain a common essential pyridoxal-5'-phosphate(PLP)/NaBH4 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-TV. 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-TV. The specific activity of K300R was reduced over 400-fold with an apparent K-m for GDP-fucose over 200 mu M. FucT-VII mutants K169R and K240R (equivalent to K228R and K300R for FucT-TV, respectively) were inactive, No change in PLP/NaBH4 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/NaBH4 inactivation,