On the reaction pathways and determination of transition-state structures for retaining alpha-galactosyltransferases

The catalytic mechanism of retaining glycosyltransferases is not yet completely understood, but one possible mechanism, by analogy with retaining glycosidases, is a double-displacement mechanism via a covalent glycosyl-enzyme intermediate (CGE). We have investigated various reaction pathways for this mechanism using non-empirical quantum mechanical methods. Because a double-displacement mechanism presumes a reaction happening in two steps, we have used predefined reaction coordinates to calculate the potential energy surface describing each step of the mechanism. By investigating several potential candidates to act as a catalytic base, this study attempts to shed some light on the unclear mechanism of the second step of the reaction. All intermediates and transition states on the reaction pathways were characterized using basis sets up to the DFT/B3LYP/6-311++G**//DFT/B3LYP/6-31G* level. Reaction pathways and structural changes were compared with the results previously obtained for inverting glycosyltransferases. The outcome of this study indicates, that among the reaction models investigated, the energetically favorable one is also the most plausible given the existing experimental data. This model requires the presence of only one catalytic acid in the active site with the UDP functioning as a general base in the second step of the reaction. This mechanism is in agreement with both kinetic data in the literature and the description of X-ray structures of retaining glycosyltransferases solved up to today.