Quantum Mechanics/Molecular Mechanics Simulations Distinguish Insulin-Regulated Aminopeptidase Substrate (Oxytocin) and Inhibitor (Angiotensin IV) and Reveal Determinants of Activity and Inhibition

Insulin-regulated aminopeptidase (IRAP) is a zinc-dependent metalloenzyme identified as a novel target for combating diabetes-induced diseases due to its crucial role in glucose metabolism and insulin sensitivity regulation. IRAP’s catalytic domain catalyzes the N-terminal peptide bond hydrolysis of natural substrate oxytocin, a neuroactive peptide linked to improved cognition and other elemental brain functions. Angiotensin IV and similar peptides are recognized as cognitive enhancers due to their ability to competitively inhibit IRAP’s proteolytic activity, thereby mitigating natural neuropeptide degradation. Despite a very similar binding complex between the substrate and the inhibitor with IRAP, particularly around the scissile bond, it is unclear why the enzyme metabolizes oxytocin but does not efficiently degrade angiotensin IV. We employed enhanced sampling quantum mechanics/molecular mechanics (QM/MM) molecular dynamics simulations and higher-level QM/MM calculations to explore the reaction of these two peptides in IRAP. The calculated energy barrier for oxytocin cleavage was in very good agreement with the experimental data. A significantly higher energy barrier for the formation of the oxyanion tetrahedral intermediate (TI) and a higher overall barrier for the peptide cleavage were observed for the reaction with angiotensin IV. Comprehensive electronic structure analysis utilizing NBO and NCI methods unveiled the molecular basis for different reactivity, a stabilizing interaction between the sigma hole of the N-terminus disulfide bond and the hybridizing lone pair of the scissile peptide nitrogen in oxytocin. The interplay between a weak noncovalent spodium bond and strong bidentate coordination of the catalytic Zn2+ by angiotensin IV caused a larger deviation of the valine C–Cα-Cβ angle from ideal tetrahedral geometry, consequently destabilizing the TI. These results underscore the critical importance of analyzing the dynamics, interactions, and electronic properties of reaction intermediates and transition states in enzymatic processes. Our findings have significant implications for the rational design and development of IRAP inhibitors as potential therapeutic agents for memory disorders, neurodegenerative diseases, and diabetes.