Hydrogen bond and close contact analyses revealed that angiotensin IV forms more stable complex with IRAP compared to oxytocin, particularly with Gly428, exhibiting a 42% higher H-bond frequency (Figure ). As a competitive inhibitor, angiotensin IV likely forms stronger interactions with the active site of IRAP, resulting in a thermodynamically more stable enzyme-inhibitor complex. This increased stability could potentially elevate the overall activation free energy barrier. To validate these hypotheses and elucidate the influence of Glu465 conformation, QM/MM umbrella sampling and adaptive string method (ASM) free energy simulations were conducted on both oxytocin and angiotensin IV complexes to model the hydrolysis reaction.