QM/MM 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 involved in the regulation of glucose metabolism and insulin sensitivity identified as a novel target for combating diabetes-induced diseases. IRAP’s catalytic domain catalyzes the N-terminal peptide bond hydrolysis of the natural substrate oxytocin, a neuroactive pep-tide 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 proteolytic activity of IRAP, reducing the degradation of natural neuropeptides. Despite a very similar binding complex between the substrate and the inhibitor with IRAP, especially around the scissile bond, it is unclear why the enzyme metabolizes oxytocin but does not efficiently degrade angiotensin IV. We employed enhanced sampling QM/MM molecular dynamics simulations to explore free energy landscapes for reaction of these two peptides in IRAP. A significantly higher energy barrier for the formation of the oxyanion tetrahedral intermediate (TI) and higher overall barrier for the peptide cleavage was observed for the reaction of angiotensin IV. Electronic structure analysis (NBO and NCI) revealed the reasons for different reactivity, including stabilization of the on the sigma hole of the N-terminus disulfide in oxytocin by the hybridizing lone pair of the scissile peptide nitrogen. The interplay between weak non-covalent spodium bond and strong bi-dentate coordination of the catalytic Zn2+ by angiotensin IV caused larger deviation of valine C-Cα-Cβ angle from the ideal tetrahedral, which destabilizes the TI. The results emphasise the importance of analysing dynamics, interactions and electronic properties of reaction intermediates and transition states in enzymes, and have implications for the de-sign and development of IRAP inhibitors for the treatment of memory disorders, neurodegenerative diseases, and diabetes.