QM/MM umbrella sampling simulations of the water nucleophilic attack. The free energy profiles exhibit significant disparities between the two peptides. A stable intermediate was observed at a reaction coordinate (OWater-CPeptide) value of 1.5 Å during the reaction with oxytocin, but not with angiotensin IV (Figure and Figure of the SI for the reactions with Glu465 in the OE1 and OE2 conformations, respectively). The simulations demonstrated that the reaction can proceed with the catalytic Glu465 in either the OE1 or OE2 conformation, with a slightly lower energy barrier for the OE1 conformation. These results indicate that oxytocin cleavage occurs via a concerted, asynchronous mechanism involving proton transfer from the water molecule to Glu465 and nucleophilic attack by hydroxide. The free energy barrier was calculated to be approximately 6.7 kcal mol -1 at a reaction coordinate value of ~1.8 Å, suggesting a late transition state (Figure , SI).