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Glycobiology Advance Access published online on June 30, 2004
Glycobiology, doi:10.1093/glycob/cwh119
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Received May 28, 2004
Revised June 24, 2004
Accepted June 25, 2004

ORIGINAL ARTICLES

Roles of active site tryptophans in substrate binding and catalysis by {alpha}-1,3 galactosyltransferase

Yingnan Zhang 1, Ashlesha Deshpande 2, Zhihong Xie 1, Ramanathan Natesh 2, K. Ravi Acharya 2, Keith Brew 1*

1 Department of Biomedical Sciences, Florida Atlantic University, Boca Raton, Florida 33341, USA
2 Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK

* To whom correspondence should be addressed. E-mail: kbrew{at}fau.edu.


  Abstract

Aromatic amino acids are frequent components of the carbohydrate binding sites of lectins and enzymes. Previous structural studies have shown that in {alpha}-1,3 galactosyltransferase, the binding site for disaccharide acceptor substrates is encircled by four tryptophans, residues 249, 250, 314 and 356. To investigate their roles in enzyme specificity and catalysis, we expressed and characterized variants of the catalytic domain of {alpha}-1,3 galactosyltransferase with substitutions for each tryptophan. Substitution of glycine for tryptophan249, whose indole ring interacts with the non-polar B face of glucose or GlcNAc, greatly increases the Km for the acceptor substrate. In contrast, the substitution of tyrosine for tryptophan314, which interacts with the {beta}-galactosyl moiety of the acceptor and UDP-galactose, decreases kcat for the galactosyltransferase reaction but does not affect the low UDP-galactose hydrolase activity. Thus, this highly conserved residue stabilizes the transition state for the galactose transfer to disaccharide but not to water. High resolution crystallographic structures of the Trp249Gly mutant and the Trp314Tyr mutant indicate that the mutations do not affect the overall structure of the enzyme or its interactions with ligands. Substitutions for tryptophan250 have only small effects on catalytic activity, but mutation of tryptophan356 to threonine reduces catalytic activity for both transferase and hydrolase activities and reduces affinity for the acceptor substrate. This residue is adjacent to the flexible C-terminus that becomes ordered on binding UDP, to assemble the acceptor binding site and influence catalysis. The results highlight the diverse roles of these tryptophans in enzyme action and the importance of kcat changes in modulating glycosyltransferase specificity.

Keywords: glycosyltransferase; mutation; crystal structure; substrate binding; catalysis.
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