Mechanistic Insights into the Hydrolysis of O-GlcNAcylation Catalyzed by Human O-GlcNAcase.

O-GlcNAc glycosylation occurs on specific serine/threonine residues of intracellular proteins, which is widely related to various diseases, including type II diabetes, cancer, and Alzheimer's disease. Human O-GlcNAcase (hOGA) is responsible for the removal of O-GlcNAc modification and thus serves as the main target for inhibitor design. In this work, we systematically investigated the mechanism catalyzed by hOGA using the quantum mechanical/molecular mechanical method. Based on calculated free energy profiles, two essential steps named cyclization (Cyc) step and ring opening step are required to generate the final hemiacetal product. The Cyc of the 2-acetamido group, the rate-limiting step, leads to the generation of the intermediate of a bicyclic oxazolinium ion (EI1). Next, the oxazoline ring could be broken via the nucleophilic attack of a water molecule at the C1 position, which generates the final product. Along with this, our simulations clearly suggest the existence of an oxazoline intermediate (EI2), which is produced via proton transfer (PT) from the 2-acetamido group (EI1) to D174. This PT step features a reversible process with a low energy barrier, which could be attributed to a low barrier hydrogen bond between the donor and acceptor. The stabilizing effect of the low barrier hydrogen bond on EI1 is proposed to be very important for accelerating the overall reaction. In fact, the site-directed mutagenesis simulations of D174A and D175A strongly indicate that the catalytic residues mainly affect the observed reaction rate by affecting the stability of the intermediate.