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

Glycobiology
Yingnan ZhangKeith Brew

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 tryptophan 249, whose indole ring interacts with the nonpolar B face of glucose or GlcNAc, greatly increases the K(m) for the acceptor substrate. In contrast, the substitution of tyrosine for tryptophan 314, which interacts with the beta-galactosyl moiety of the acceptor and UDP-galactose, decreases k(cat) 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 Trp(249)Gly mutant and the Trp(314)Tyr mutant indicate that the mutations do not affect the overall structure of the enzyme...Continue Reading

Citations

Nov 9, 2010·Nature Biotechnology·Carlos J BosquesGanesh Venkataraman
Mar 2, 2005·Nature Reviews. Microbiology·Christine M Szymanski, Brendan W Wren
Jul 23, 2009·The Journal of Biological Chemistry·Percy Tumbale, Keith Brew
Oct 24, 2006·Nature Chemical Biology·Luke L LairsonStephen G Withers
Dec 19, 2014·Magnetic Resonance in Medicine : Official Journal of the Society of Magnetic Resonance in Medicine·Benjamin B BartelleDaniel H Turnbull
Jun 3, 2009·Biochemical and Biophysical Research Communications·Haryati JamaluddinK Ravi Acharya
Apr 24, 2012·Carbohydrate Research·Hansel GómezLaura Masgrau
Jan 4, 2017·Biochemistry Research International·Eliane EvanovichMaria Lúcia Harada

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