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

Abstract

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.

QM/MM Studies Reveal How Substrate–Substrate and Enzyme–Substrate Interactions Modulate Retaining Glycosyltransferases Catalysis and Mechanism

“…Nevertheless, the effect of having a Mn 2+ cation instead of the modeled Mg 2+ on the chemical step itself was tested for the LgtC system (Gómez, Polyak, et al, 2012). The calculated reaction mechanism is the same for both metal ions, with the potential energy barriers being slightly different ($1.5 kcal/mol lower for Mn 2+ , thus, with Mn 2+ the reaction is somewhat faster), which is in agreement with recent studies on the retaining α-1,2-mannosyltransferase Krep2p/Mnt1p (Bobovská et al, 2014) and GalNAc-T2 (Trnka et al, 2015).…”

“…What is clear, though, is that an asynchronous front-side mechanism is active in this enzyme (with bond dissociation and formation taking place one after the other). Later studies of this enzyme seem to indicate that a short-lived ion pair intermediate may be actually formed (Lira-Navarrete et al, 2014;Trnka et al, 2015). The calculated stationary points reported in our work are depicted in Fig.…”

“…The QM/MM calculations carried out by our group (and by others) on retaining GTs have had an important role in characterizing the front-side attack mechanism as the general one followed by these enzymes (at least for the studied systems, and only with the possible exception of family GT6 members) (Ardèvol & Rovira, 2011;Bobovská et al, 2014Bobovská et al, , 2015Gómez et al, 2013;Gómez, Polyak, et al, 2012;Gómez et al, 2014;Lira-Navarrete et al, 2014;Rojas-Cervellera et al, 2013;Trnka et al, 2015). Crystallographic structural data with substrate analogues or product mimics also support this as the preferred mechanism.…”

“…In fact, only one transition state could be found by unrestrained TS search optimization, but it could not be fully characterized by frequency calculations. Thus, the topology of the PES in this region, together with the limitations of the density functional method used to describe it (Trnka et al, 2015), did not allow us to completely describe the type of front-side mechanism used by GalNAc-T2 (Fig. 1B vs. C).…”

“…In the past years, hybrid quantum mechanics/molecular mechanics (QM/MM) calculations have proved to be very valuable in the study of retaining GTs mechanism (Ardèvol & Rovira, 2011;Bobovská, Tvaroška, & Kóňa, 2014Gómez, Polyak, Thiel, Lluch, & Masgrau, 2012;Gómez et al, 2014;Lira-Navarrete et al, 2014;Rojas-Cervellera, Ardèvol, Boero, Planas, & Rovira, 2013;Trnka, Kozmon, Tsvaroska, & Koca, 2015). Most of the systems for which a crystallographic structure was available have been studied theoretically and much insight has been gained in the description and understanding of how these enzymes catalyze the formation of a new glycosidic bond.…”