QM/MM study on catalytic mechanism of aspartate racemase from Pyrococcus horikoshii OT3

The enzyme aspartate racemase from Pyrococcus horikoshii OT3 catalyzes the interconversion between L- and D-Asp. In this work, we employed the hybrid QM/MM approach with the self-consistent charge-density functional tight binding (SCC-DFTB) model to study the catalytic mechanism for the conversion of L-Asp into D-Asp. The molecular dynamics simulation showed that the substrate L-Asp forms an extensive network of interactions with the active-site residues of the aspartate racemase through its side chain carboxylate, ammonium group, and a-carboxylate. The potential of mean force calculations confirmed that the racemization reaction involves two proton transfers (from the alpha-carbon to Cys194 and from Cys82 to the alpha-carbon), which occurs in a concerted way, although highly asynchronous. The calculated free energy of activation is 17.5 kcal/mol, which is consistent with the reaction rate measured from experiment. An electrostatic interaction analysis was performed to estimate the key role played by individual residues in stabilizing the transition state. The docking study on the binding of L-Asp and D-Asp to aspartate racemase indicates that this enzyme employs a "two-base'' mechanism not a "one-base'' mechanism.