The classical simulations for the H134L mutant clearly demonstrate the formation of a new water route that may be responsible for the ATPase activity, therefore, to validate any role of water in ATPase activity, we performed the reaction kinetics using QM/MM calculations. In so doing, we performed the clustering of the MD trajectories and selected a representative snapshot from the most populated trajectory (Figure ). This snapshot has further proceeded for the QM/MM calculations. Interestingly, in most of the MD trajectory of the simulations, two water molecules, W1 and W2, were consistently close to the catalytic site. One of these water molecules is stabilized by a strong hydrogen bond with N141. We believe that these two water molecules, along with N141, may be crucial for the ATPase activity, and therefore, we included these residues in the QM region during the QM/MM calculations. After optimization of the reactant geometry, we performed a potential energy surface (PES) scan to obtain the reaction profile and the activation of the reaction (see Figure ). During the PES scanning, we found that the reaction is triggered by the transfer of a proton from attacking water W1 to the oxygen of terminal phosphate via W2, which leave an OH -on W1. Subsequently, the OH -performs a nucleophilic attack on the γ-Phosphate of ATP, which initiates the cleavage of the P-O bond of the terminal phosphate of ATP and forms the ADP and the phosphate as an ultimate product. Interestingly, the reaction barrier for the overall process is 22.4 kcal/mol, which is in the enzymatic range. In summary, the present study provides new insight into the mechanism of homoserine kinase. In contrast to the previous consensus that the phosphorylation of the L-homoserine is substrate assisted process, we reveal that the role of Histidine is crucial and acts as a base during the reaction. Interestingly, our proposed catalytic mechanism is in good agreement with sitedirected mutagenesis experiments where the mutation of the H138L decreases the native activity and causes the emergence of the secondary function of ATPase activity. The present study also highlights the mechanism of functional promiscuity due to a single-site mutation.