Glycobiology Advance Access originally published online on July 31, 2009
Glycobiology 2009 19(11):1224-1234; doi:10.1093/glycob/cwp114
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Comparative study of substrate and product binding to the human ABO(H) blood group glycosyltransferases
2 Alberta Ingenuity Centre for Carbohydrate Science and Department of Chemistry, University of Alberta, Edmonton, Alberta, T6G 2G2 Canada
3 Carlsberg Laboratory, Gamle Carlsberg Vej 10, 2500 Valby, Denmark
1 To whom correspondence should be addressed: Tel: (780) 492-3501; Fax: (780) 492-8231; e-mail: john.klassen{at}ualberta.ca
Received on March 7, 2009; revised on July 20, 2009; accepted on July 21, 2009
The first comparative thermodynamic study of the human blood group glycosyltransferases, 
-(1
3)-N-acetylgalactosaminyltransferase (GTA) and -(13)-galactosyltransferase (GTB), interacting with donor substrates, donor and acceptor analogs, and trisaccharide products in vitro is reported. The binding constants, measured at 24°C with the direct electrospray ionization mass spectrometry (ES-MS) assay, provide new insights into these model GTs and their interactions with substrate and product. Notably, the recombinant forms of GTA and GTB used in this study are shown to exist as homodimers, stabilized by noncovalent interactions at neutral pH. In the absence of divalent metal ion, neither GTA nor GTB exhibits any appreciable affinity for its native donors (UDP-GalNAc, UDP-Gal). Upon introduction of Mn2+, both donors undergo enzyme-catalyzed hydrolysis in the presence of either GTA or GTB. Hydrolysis of UDP-GalNAc in the presence of GTA proceeds very rapidly under the solution conditions investigated and a binding constant could not be directly measured. In contrast, the rate of hydrolysis of UDP-Gal in the presence of GTB is significantly slower and, utilizing a modified approach to analyze the ES-MS data, a binding constant of 2 x 104 M–1 was established. GTA and GTB bind the donor analogs UDP-GlcNAc, UDP-Glc with affinities similar to those measured for UDP-Gal and UDP-GalNAc (GTB only), suggesting that the native donors and donor analogs bind to the GTA and GTB through similar interactions. The binding constant determined for GTA and UDP-GlcNAc (
1 x 104 M–1), therefore, provides an estimate for the binding constant for GTA and UDP-GalNAc. Binding of GTA and GTB with the A and B trisaccharide products was also investigated for the first time. In the absence of UDP and Mn2+, both GTA and GTB recognize their respective trisaccharide products but with a low affinity 103 M–1; the presence of UDP and Mn2+ has no effect on A trisaccharide binding but precludes B-trisaccharide binding.
Key words: association constants / electrospray ionization mass spectrometry / human ABO(H) blood group glycosyltransferases / protein dimerization / protein-oligosaccharide complexes