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 (ΔH_{a , ΔSa ) decrease significantly. However, the free energy of association (ΔGa ) is unchanged at physiological temperature. The differences in the ΔHa and ΔSa 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.}