Human α1,3/4-fucosyltransferases -: I.: Identification of amino acids involved in acceptor substrate binding by site-directed mutagenesis

In a previous study (Xu, Z., Vo, L., and Macher, B. A. (1996) J. Biol Chem. 271, 8818-8823), a domain swapping approach demonstrated that a region of amino acids found in human alpha 1,3/4-fucosyltransferase III (FucT III) conferred a significant increase in alpha 1,4-FucT acceptor substrate specificity into alpha 1,3-fucosyltransferase V (FucT V), which, under the same assay conditions, has extremely low alpha 1,4-FucT acceptor substrate specificity. In the current study, site-directed mutagenesis was utilized to identify which of the eight amino acids, associated with alpha 1,4-FucT acceptor substrate specificity, is/are responsible for conferring this new property. The results demonstrate that increased alpha 1,4-FucT activity with both disaccharide and glycolipid accepters can be conferred on FucT V by modifying as few as two (Asn(86) to His and Thr(87) to Ile) of the eight amino acids originally swapped from FucT III into the FucT V sequence. Neither single amino acid mutant had increased alpha 1,4-FucT activity relative to that of FucT V. Kinetic analyses of FucT V mutants demonstrated a reduced K-m for Gal beta 1,3GlcNAc (type 1) acceptor substrates compared with native FucT V. However, this was about 20-fold higher than that found for native FucT III, suggesting that other amino acids in FucT III must contribute to its overall binding site for type 1 substrates, These results demonstrate that amino acid residues near the amino terminus of the catalytic domain of FucT In: contribute to its acceptor substrate specificity.