At. Nguyen et al., HUMAN ALPHA-1,3 4-FUCOSYL-TRANSFERASES - I - IDENTIFICATION OF AMINO-ACIDS INVOLVED IN ACCEPTOR SUBSTRATE-BINDING BY SITE-DIRECTED MUTAGENESIS/, The Journal of biological chemistry, 273(39), 1998, pp. 25244-25249
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 II
I (FucT III) conferred a significant increase in alpha 1,4-FucT accept
or substrate specificity into alpha 1,3-fucosyltransferase V (FucT V),
which, under the same assay conditions, has extremely low alpha 1,4-F
ucT acceptor substrate specificity. In the current study, site-directe
d 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 gly
colipid accepters can be conferred on FucT V by modifying as few as tw
o (Asn(86) to His and Thr(87) to Ile) of the eight amino acids origina
lly swapped from FucT III into the FucT V sequence. Neither single ami
no 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 n
ative FucT V. However, this was about 20-fold higher than that found f
or native FucT III, suggesting that other amino acids in FucT III must
contribute to its overall binding site for type 1 substrates, These r
esults demonstrate that amino acid residues near the amino terminus of
the catalytic domain of FucT In: contribute to its acceptor substrate
specificity.