The tetrahedral intermediate (TI) formed in the reaction with both peptides is predominantly stabilized by the oxyanion hole, comprised of the hydrogen bond-donating hydroxyl group of Tyr549. The scissile peptide bond oxygen atom, which develops a partial negative charge during hydroxide attack, is further stabilized by the positively charged Zn 2+ ion. Notably, the intermediate along the oxytocin reaction pathway starting from Glu465 in the OE2 conformation demonstrated the potential to adopt an alternative hydrogen bonding network, which had minimal effects on the calculated potential of mean force (PMF) (see Figure of the SI). Conversely, simulations of the angiotensin IV reaction revealed that proton transfer from the zinc-bound water to Glu465 in either the OE1 and OE2 conformation yielded a higher energy intermediate, with no distinct free energy well corresponding to the tetrahedral intermediate (Figure and Figure in the SI).