Human alpha1,3/4-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 alpha1, 3/4-fucosyltransferase III (FucT III) conferred a significant increase in alpha1,4-FucT acceptor substrate specificity into alpha1, 3-fucosyltransferase V (FucT V), which, under the same assay conditions, has extremely low alpha1,4-FucT acceptor substrate specificity. In the current study, site-directed mutagenesis was utilized to identify which of the eight amino acids, associated with alpha1,4-FucT acceptor substrate specificity, is/are responsible for conferring this new property. The results demonstrate that increased alpha1,4-FucT activity with both disaccharide and glycolipid acceptors can be conferred on FucT V by modifying as few as two (Asn86 to His and Thr87 to Ile) of the eight amino acids originally swapped from FucT III into the FucT V sequence. Neither single amino acid mutant had increased alpha1,4-FucT activity relative to that of FucT V. Kinetic analyses of FucT V mutants demonstrated a reduced Km for Galbeta1,3GlcNAc (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 III contribute to its acceptor substrate specificity.
Nguyen
A T
AT
Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, California 94132, USA.
Holmes
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EH
Whitaker
J M
JM
Ho
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S
Shetterly
S
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Macher
B A
BA
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers; D005647:Fucosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity
D000596:Amino Acids; D017931:DNA Primers; D005647:Fucosyltransferases; C019818:3-galactosyl-N-acetylglucosaminide 4-alpha-L-fucosyltransferase