SPECIFICITY STUDIES OF THE GDP-[L]-FUCOSE - 2-ACETAMIDO-2-DEOXY-BETA-[D]-GLUCOSIDE (FUC-]ASN-LINKED GLCNAC) 6-ALPHA-FUCOSYL-TRANSFERASE FROM RAT-LIVER GOLGI MEMBRANES

The specificity of Golgi-membrane glycoprotein 6-alpha-[L]-fucosyltran sferase [GDP-[L]-fucose: 2-acetamido-2-deoxy-beta-[D]-glucoside (Fuc - -> Asn-linked GlcNAc) 6-alpha-[L]-fucosyltransferase; EC 2.4.1.68] has been assessed with regard to substrate covalent structures and the ef fect of a protein matrix on the conformational display of those covale nt structures. Specificity was studied by direct comparison of the sub strate quality of nine 6-biotinamidohexanoylAsn (=R) derivatives of in termediates and products in the pathway from Man(5)GlcNAc(2)-R to a fu lly sialylated biantennary complex-type glycan. The Man, derivative an d the sialic acid-containing glycans were completely inactive as subst rates. The other glycans were all fucosylated; the best substrate was GlcNAcMan(3),GlcNAc(2)-R. The protein-matrix effect was studied by com paring the substrate quality of the same 6-biotinamidohexanoylAsn deri vatives as well as the corresponding biotinylAsn derivatives free in s olution and bound to streptavidin. On the basis of a model derived fro m the known 3D structure of biotin (biocytin)-saturated streptavidin, it was predicted that the fucosylation site in the substrates would be completely masked in the biotin-binding pocket in the biotinyl deriva tives (proximal display), and at least partially masked in the 6-bioti namidohexanoyl derivatives (distal display). The activity measurements were in agreement with these predictions; the glycan structures GlcNA cMan(5)GlcNAc(2)-, GlcNAcMan(3)GlcNAc(2)-, and GlcNAc,Man,GlcNAc(2)- w ere readily fucosylated as derivatives free in solution, but were tota lly inert in the proximal complex with streptavidin. In the distal com plexes the latter two structures were found to be fucosylated very slo wly while the former structure was inactive.