Ab Initio Molecular Orbital Study of the Catalytic Mechanism of Glycosyltransferases:  Description of Reaction Pathways and Determination of Transition-State Structures for InvertingN-Acetylglucosaminyltransferases

The catalytic mechanism of the GlcNAc transfer by inverting N -acetylglucosaminyltransferases is explored for the first time using ab initio quantum chemical methods. The structural model describing the active site where the reaction occurs consists of all essential molecules or their fragments assumed to be involved in the mechanism: a complete sugar-donor molecule, UDP-GlcNAc, a hydroxyl group of the oligosaccharide-acceptor modeled by methanol, a divalent metal cofactor represented by Mg 2+, as well as the essential parts of the catalytic acid (A) and catalytic base (B) modeled by acetic acid and acetate molecules. Different possible mechanisms of reaction have been followed by means of several two-dimensional potential energy maps calculated as a function of predefined reaction coordinates. Potential energy surfaces calculated at the HF/6-31G* level revealed 11 transition states and five intermediates associated with distinct possible reaction pathways. All stationary points, transition states, and intermediates, were characterized at HF/6-31G*, HF/6-31++G**//HF/6-31G*, DFT/B3LYP/6-31G*, and DFT/B3LYP/6-31++G**//DFT/B3LYP/6-31G* levels. A detailed description of the reaction pathways that includes energetic evaluations and the structural modifications of the different participants occurring along the catalytic process is given, followed by a discussion on their feasibility, consequences, and implications for the catalytic mechanism. Among the different reaction pathways, a stepwise reaction pathway assuming the enrolment of only a catalytic base appeared to be the most probable reaction path and consistent with the existing experimental data.