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.
Highlights
Protein glycosylation is known to play a pivotal role in many aspects of protein biochemistry, and there have been many examples where carbohydrate structures carry out a significant biological function. [1,2,3] Glycans exist in a vast array of diverse structures built up from just a few small basic fragments
We have shown that human isoform 2 of the polypeptide UDP-GalNAc transferase catalyses a same-face nucleophilic substitution with internal return (SNi)
The optimized transition state for the reaction is 13.8 kcal mol−1 higher in energy than the reactant, while the energy of the product complex is 6.7 kcal mol−1 lower. This corresponds to a dissociated oxocarbenium state just before its nucleophilic capture by the acceptor threonine oxygen
Summary
Protein glycosylation is known to play a pivotal role in many aspects of protein biochemistry, and there have been many examples where carbohydrate structures (glycans) carry out a significant biological function. [1,2,3] Glycans exist in a vast array of diverse structures built up from just a few small basic fragments. [1,2,3] Glycans exist in a vast array of diverse structures built up from just a few small basic fragments. This can be directly compared to the protein world, constructed purely from simple amino acids. [1] The so-called glycocode is just implicitly present in the regulation of hundreds of different highly specialized enzymes, glycosidases and glycosyltransferases, forming the glycosylation cascade. For this reason, understanding the reactivity of glycosyltransferases is essential to being able to decode the glycocode. A lot of scientific attention has been recently focused on this issue in an attempt to determine the reaction mechanism of retaining glycosyltransferases, with mixed results. [4, 5]
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