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Comparative study of substrate and product binding to the human ABO(H) blood group glycosyltransferases

  • Author(s) / Creator(s)
  • The first comparative thermodynamic study of the human blood group glycosyltransferases, α-(1→3)-N-acetylgalactosaminyltransferase (GTA) and α-(1→3)-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 × 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 × 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.

  • Date created
    2009
  • Subjects / Keywords
  • Type of Item
    Article (Published)
  • DOI
    https://doi.org/10.7939/R3V11VZ64
  • License
    © 2009 Soya, N., Shoemaker, G. K., Palcic, M. M., & Klassen, J. S. This version of this article is open access and can be downloaded and shared. The original author(s) and source must be cited.
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  • Citation for previous publication
    • Soya, N., Shoemaker, G. K., Palcic, M. M., & Klassen, J. S. (2009). Comparative study of substrate and product binding to the human ABO(H) blood group glycosyltransferases. Glycobiology, 19(11), 1224-1234. http://doi.org/10.1093/glycob/cwp114
  • Link to related item
    http://doi.org/10.1093/glycob/cwp114