The catalytic mechanism of retaining glycosyltransferases remains a controversial issue in glycobiology. It was first suggested to be that of a double-displacement mechanism by analogy to the well-established mechanism of retaining glycosidases. However,many GTs do not have a putative nucleophile protein residue. This prompted some authors to suggest an unusual front side single displacement (SNi-like mechanism), a feasible mechanism as shown by both theoretical (1) and experimental (2) studies.
But other GTs, such as family GT6 mammalian α3-galactosyltransferase (α3GalT) and blood group A and B glycosyltransferase (GTA/GTB), do have a putative nucleophilic residue properly located in the active site to participate in catalysis. We now demonstrate by QM/MM metadynamics simulations that α3GalT operates via a double-displacement mechanism, with the formation of a glycosyl-enzyme covalent intermediate. This result complements previous experimental evidences: the chemical rescue of an inactive nucleophile mutant (E317A) in α3GalT (3), and the trapping of a transient covalent intermediate in a cysteine mutant (E303C) of blood group GTA and GTB (4).
We now conclude that both mechanisms are at play depending on the enzyme. Their main difference is the way the enzyme stabilizes the oxocarbenium ion-like species. In GTs lacking a catalytic nucleophile, the electrostatic potential at the active site is such that it can stabilize the oxocarbenium ion-like intermediate for a very short time, but long enough for the active site to reorganize. In the case of family 6 GTs, the oxocarbenium ion-like transition state is stabilized by the formation of a covalent bond with a carboxylate residue (catalytic nucleophile). Therefore, both modes of operation can be considered as variations of a unique mechanism.
1. Ardèvol, A., Rovira, C.Angew. Chem. Int. Ed.2011, 50, 10897.
2. Lee, et al.Nat. Chem. Biol.2011, 7, 631.
3. Monegal, A., Planas, A.J. Am. Chem. Soc.2006, 128, 16030.
4. Soya N, et al.Glycobiology.2011, 21, 547.
