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

Affinities of the human blood group glycosyltransferases, alpha-(1-->3)-N-acetylgalactosaminyltransferase (GTA) and alpha-(1-->3)-galactosyltransferase (GTB) for their common acceptor substrate alpha-l-Fucp-(1-->2)-beta-d-Galp-O(CH2)(7)CH3 (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 Mn(2+) 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 (DeltaH(a), DeltaS(a)) decrease significantly. However, the free energy of association (DeltaG(a)) is unchanged at physiological temperature. The differences in the DeltaH(a) and DeltaS(a) 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.