Temperature-dependent cooperativity in donor-acceptor substrate binding to the human blood group glycosyltransferases

Abstract

Affinities of the human blood group glycosyltransferases, α-(1→3)- N -acetylgalactosaminyltransferase (GTA) and α-(1→3)-galactosyltransferase (GTB) for their common acceptor substrate α-l-Fucp-(1→2)-β-d-Galp-O (CH2) 7CH3 (1), in the absence and presence of bound uridine 5′-diphosphate (UDP) and Mn2+ were determined using temperature-controlled electrospray ionization mass spectrometry. The presence of bound UDP and Mn2+ in the donor binding site has a marked influence on the thermodynamic parameters for the association of 1 with GTA and GTB. Both the enthalpy and entropy of association (Δ Ha, Δ Sa) decrease significantly. However, the free energy of association (Δ Ga) is unchanged at physiological temperature. The differences in the Δ Ha and Δ Sa values determined in the presence and absence of bound UDP are attributed to structural changes in the glycosyltransferases induced by the simultaneous binding of 1 and UDP. © The Author 2008. Published by Oxford University Press. All rights reserved.