Stepwise Catalytic Mechanism via Short-Lived Intermediate Inferred from Combined QM/MM MERP and PES Calculations on Retaining Glycosyltransferase ppGalNAcT2

The glycosylation of cell surface proteins plays a crucial role in a multitude of biological processes, such as cell adhesion and recognition. To understand the process of protein glycosylation, the reaction mechanisms of the participating enzymes need to be known. However, the reaction mechanism of retaining glycosyltransferases has not yet been sufficiently explained. Here we investigated the catalytic mechanism of human isoform 2 of the retaining glycosyltransferase polypeptide UDP-GalNAc transferase by coupling two different QM/MM-based approaches, namely a potential energy surface scan in two distance difference dimensions and a minimum energy reaction path optimisation using the Nudged Elastic Band method. Potential energy scan studies often suffer from inadequate sampling of reactive processes due to a predefined scan coordinate system. At the same time, path optimisation methods enable the sampling of a virtually unlimited number of dimensions, but their results cannot be unambiguously interpreted without knowledge of the potential energy surface. By combining these methods, we have been able to eliminate the most significant sources of potential errors inherent to each of these approaches. The structural model is based on the crystal structure of human isoform 2. In the QM/MM method, the QM region consists of 275 atoms, the remaining 5776 atoms were in the MM region. We found that ppGalNAcT2 catalyzes a same-face nucleophilic substitution with internal return (SNi). The optimized transition state for the reaction is 13.8 kcal/mol higher in energy than the reactant while the energy of the product complex is 6.7 kcal/mol lower. During the process of nucleophilic attack, a proton is synchronously transferred to the leaving phosphate. The presence of a short-lived metastable oxocarbenium intermediate is likely, as indicated by the reaction energy profiles obtained using high-level density functionals.Author Summary: Cell surface proteins are covered by a diverse array of glycan structures, important for mutual cell recognition and communication. These glycans are complex branched molecules assembled from monosaccharide units by a sophisticated cascade of enzymes from the group of glycosyltransferases. Disruptions in the synthesis of glycans are linked to various diseases with the most prominent example being cancer. To understand or control the process of glycosylation, the reaction mechanisms of the participating enzymes need to be known. Here we investigate the catalytic mechanism of human glycosyltransferase ppGalNAcT2 using the tools of computational chemistry. By modelling the crucial parts of the enzyme using a quantum mechanics-based description, we are able to trace the whole reaction path leading from the reactant state to the product state. Our results provide a reliable description of the motion of all important atoms during the reaction and they are fully consistent with available experimental data. The insights obtained in this study can be further used to design a potent inhibitor molecule, usable as a potential drug for diseases involving increased activity of the enzyme.