Computational evidence for the substrate-assisted catalytic mechanism of O-GlcNAcase. A DFT investigation

Abstract

A DFT computational investigation of the catalytic mechanism of O-GlcNAcase shows the existence of a substrate-assisted reaction pathway similar to that proposed in the literature on the basis of experimental evidence: the carbonyl oxygen of the N-acyl group bonded at C2 of the substrate pyranose ring attacks the anomeric carbon affording a bicyclic oxazoline intermediate and causing the breaking of the glycosidic bond and the expulsion of the aglycon. This occurs in a single kinetic step where the transfer of a proton from Asp-243 (behaving as a general base) to the leaving group is simultaneous to the cycle formation and departure of the aglycon (an activation barrier E_a of 16.5 kcal mol⁻¹). Even if the other component of the catalytic dyad (Asp-242) is not actually involved in a proton transfer (as commonly suggested), it plays an important role in the catalysis through a complex network of hydrogen bonds that contribute to lower the activation barrier. The transition state of the process resembles an oxocarbenium ion (half chair conformation with an approximately planar sp² anomeric carbon). Following the lines of previous experiments aimed to demonstrate the existence of a substrate-assisted mechanism, it is found that the computed E_a increases when the hydrogen atoms of the N-acetyl group are replaced with one, two and three F atoms and that a good linear correlation exists between the activation barrier E_a and the σ* Taft electronic parameter of the fluoro-substituted N-acetyl groups.