HUMAN ALPHA-1,3 4-FUCOSYL-TRANSFERASES - III - A LYS/ARG RESIDUE LOCATED WITHIN THE ALPHA-1,3-FUCT MOTIF IS REQUIRED FOR ACTIVITY BUT NOT SUBSTRATE-BINDING/

Amino acid sequence alignment of human alpha 1,3/4-fucosyltransferases (FucTs) demonstrates that three highly conserved Lys residues are pre sent in the catalytic domain of FucTs III, TV, V, and VI. Two of these sites are conserved in FucT VII, with the third located within the al pha 1,3-FucT motif as a conservative change to Arg at position 223. Si te-directed mutagenesis experiments were conducted to change Lys(255) Of FucT V (equivalent to Arg(223) of FucT VII) to either Arg(255) or A la(255). Enzyme assays demonstrate that the FucT V K255R mutant has a 34-fold lower specific activity than native FucT V and that the K255A mutant is inactive. Site-directed mutagenesis of FucT VII was also con ducted to change Arg(223) to Lys(223) for analysis of the effect on en zyme kinetic parameters. No differences in acceptor specificities or K -m values for either substrate were observed between native FucT VII a nd the R223K mutant; however, the purified R223K mutant enzyme had a 2 -fold increased specific activity compared with purified native FucT V II. No change in GDP-fucose-protectable pyridoxal-P/NaBH4 inactivation was observed for native or mutant FucT V or VII, further supporting t he absence of involvement of this residue in sugar nucleotide binding. The results indicate that a basic residue in this position is require d for enzyme activity, with a Lys residue providing higher intrinsic a ctivity. The lack of influence of this site on substrate binding param eters and its location within the alpha 1,3-FucT motif suggest that at least some of the residues within this motif are involved in catalysi s rather than substrate binding.