O-GlcNAcylation is an essential and dynamic post-translational modification that is regulated by only two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA)[1]. O-GlcNAc modifications are catalysed by OGT and are removed by the antagonistic enzyme OGA. The mechanisms by which OGT recognizes diverse substrates and the functional variation, together with the mechanisms underlying OGT catalysis remains to be unclear. While the reported structures of OGT are very similar, at least three different mechanisms have been proposed which differ in the identity of the general base[2,3]. For α-phosphate moiety of UDP-GlcNAc, one of the proposed catalytic base, its pKa is heavily debated in the literature[2]. Using quantum chemical calculations together with continuum electrostatics models, we have successfully established the pKa of the α-phosphate moiety of UDP-GlcNAc. Also there is an uncertainty on the preferred conformation of serine side chain, which acts as an acceptor in the OGT catalytic mechanism. To address this, we have used free energy calculations to establish the preferred conformer of serine side chain in the protein from the three stable rotamers captured by X-ray structures (+gauche, -gauche and trans). The current studies will provide a basis for further computational studies of the OGT catalysis.