QM/MM study of the catalytic mechanism of GalNAc removal from GM2 ganglioside catalyzed by human β-hexosaminidaseA.

This work is based on the glycosidase β-hexosaminidase, in particular, on β-HexA (HexA), involved in the emergence of the Sandoff disease. Its function is to cleave the N-acetylgalactosamine (GalNAc) or N-acetylglucosamine moieties from its substrates. Here we reveal and consolidate many important aspects of the catalytic mechanism of GalNAc removal from the GM2 ganglioside. The reaction mechanism for this reaction is proposed to be substrate-assisted. It is expected to evolve through a mechanism similar to others glycosidase mechanisms, even though this fact has never been confirmed and the proposal still lacks atomic level detail in its description. To overcome these two limitations we have used the ONIOM formalism (B3LYP:Amber and M06-2X:Amber) with electrostatic embedding to calculate a bidimensional potential energy surface for the rate-limiting step. The potential energy surface reveals the mechanism with atomic detail. The formation of the covalent bond within the substrate has been confirmed, and its energy of stabilization has been calculated as 5.1 kcal/mol. The electronic energy barrier for this step is 22.5 kcal/mol at the hybrid ONIOM(M06-2X:Amber) level, well in line with the typical experimental values for similar reactions in glycosylases. The conformational transitions of the substrate GalNAc ring have been mapped in the PES for the first time. They support the hypothesis of a (4)S(2) skew boat-(4)E envelope-(4)C(1) chair pathway and provide much finer detail to the earlier proposals. In general the results are in line with earlier predictions.