The conformational flexibility of the catalytic Glu465 and peptide interactions with the GAMEN loop were identified as critical determinants of peptide reactivity. The reaction mechanism involves a two-step proton transfer process, initiated by the Zn 2+ -bound water molecule. The OE1 conformation of Glu465 was found to facilitate proton transfer to the scissile nitrogen in the second reaction step. Unrestrained simulations revealed superior hydrogen bonding of angiotensin IV within the IRAP active site, particularly with Gly428 and Ala429 of the GAMEN loop. This tighter complex formation contributes to an increased activation energy barrier for angiotensin IV hydrolysis.