Acceptor hydroxyl group mapping for human milk alpha 1-3 and alpha 1-3/4 fucosyltransferases

Two different fucosyltransferases (Fuc-Ts) have been isolated from human milk, an alpha 1-3 Fuc-T and an alpha 1-3/4 Fuc-T, for mapping of their acceptor binding sites. Kinetic studies employing a series of monodeoxygenated and modified Gal beta 1-->4Glc-NAc beta OR and Gal beta 1-->3GlcNAc beta OR acceptor substrates showed that modifications are tolerated at every hydroxyl group in these substrates except for 6-OH of galactose and 3- or 4-OH of N-acetylglucosamine. Deoxygenation at these positions rendered these compounds inactive as both substrates and inhibitors. These essential hydroxyl groups, which are required for recognition of the substrates, are identical to the key polar groups that have previously been reported for cloned FucTs III, IV and V.