Glycobiology Advance Access originally published online on June 30, 2004
Glycobiology 2004 14(12):1295-1302; doi:10.1093/glycob/cwh119
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Glycobiology vol. 14 no. 12 © Oxford University Press 2004; all rights reserved.
Roles of active site tryptophans in substrate binding and catalysis by 
-1,3 galactosyltransferase
5 Department of Biomedical Sciences, Florida Atlantic University, Boca Raton, FL 33341; 6 Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK
Received on May 28, 2004; revised on June 24, 2004; accepted on June 25, 2004
Aromatic amino acids are frequent components of the carbohydrate binding sites of lectins and enzymes. Previous structural studies have shown that in 
-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 -1,3 galactosyltransferase with substitutions for each tryptophan. Substitution of glycine for tryptophan 249, whose indole ring interacts with the nonpolar B face of glucose or GlcNAc, greatly increases the Km for the acceptor substrate. In contrast, the substitution of tyrosine for tryptophan 314, which interacts with the ß-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 tryptophan 250 have only small effects on catalytic activity, but mutation of tryptophan 356 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.
1 Present address: Department of Protein Engineering, Genentech, 1 DNA Way, South San Francisco, CA 94080
2 Present address: CDFD, Hyderabad 500 076, India
3 Present address: ICGEB, Aruna Asaf Ali Marg, New Delhi 110 067, India
4 To whom correspondence should be addressed; e-mail: kbrew{at}fau.edu
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