STRUCTURE-FUNCTION ANALYSIS OF HUMAN ALPHA-1-]3FUCOSYLTRANSFERASES - A GDP-FUCOSE-PROTECTED, N-ETHYLMALEIMIDE-SENSITIVE SITE IN FUCT-III AND FUCT-V CORRESPONDS TO SER(178) IN FUCT-IV

Human alpha 1-->3fucosyltransferases constitute a family of closely related membrane-bound enzymes distinguished by differences in acceptor specificities and inherent protein biochemical properties. One such biochemical property is sensitivity to enzyme inactivation by sulfhydral-group modifying reagents such as N-ethylmaleimide. The basis for this property has been studied using a fusion protein of FucT-III anf FucT-V composed of protein A coupled to the catalytic domain of the enzyme. The results indicate that modification of FucT-V by 5,5'-dithiobis(2-nitrobenzoic acid) resulted in efficient enzyme inactivation that could be reversed by excess thiol reagent suggesting that the free sulfhydral group on the enzyme was required for activity. Recombinant forms of both FucT-III and FucT-V were irreversibly inactivated by N-ethylmaleimide and could be effectively protected from inactivation by GDP-fucose and GDP but not by UDP-galactose, fucose, or N-acetyllactosamine. Analysis of the distribution of Cys residues in aligned sequences of cloned human alpha 1-->3fucosyltransferases indicated one site, Cys(143) of FucT-III and Cys(156) of FucT-V, corresponded to the highly conservative replacement of Ser(178) in FucT-IV, an enzyme insensitive to N-ethylmaleimide. A site-directed mutagenesis experiment was performed to replace Ser(178) of FucT-IV with a Cys residue. The mutant FucT-IV enzyme was active; however, the K-m for GDP-fucose was increased about 3-fold compared to the native enzyme to 28 +/- 3 mu M. This enzyme was N-ethylmaleimide sensitive and could be partially protected by GDP-fucose but not N-acetyllactosamine. These results support the importance of Ser(178) of FucT-IV in donor substrate binding and strongly suggest analogous Cys residues are the GDP-fucose protectable, N-ethylmaleimide-sensitive sites present in FucT-III and -V.