Why Bestatin Prefers\nHuman Carnosinase 2 (CN2) to\nHuman Carnosinase 1 (CN1)

Human carnosinases (CNs) are Xaa-His metal-ion-activated\naminopeptidases\nthat break down bioactive carnosine and other histidine-containing\ndipeptides. Carnosine is a bioactive peptide found in meat and prevalently\nused as a supplement and in functional food formulation. Nonetheless,\ncarnosine is digested by CNs rapidly after ingestion. CNs have two\nisoforms (carnosinase 1 (CN1) and carnosinase 2 (CN2)), where CN1\nis the main player in carnosine digestion. CNs contain a catalytic\nmetal ion pair (Zn<sup>2+</sup> for CN1 and Mn<sup>2+</sup> for CN2)\nand two subpockets (S1 and S1′ pockets) to accommodate a substrate.\nBestatin (BES) has been reported to be active for CN2; however, its\ninhibition ability for CN1 has remained under debate, because the\nunderlying mechanism remains unclear. This information is important\nfor designing novel CN1-selective inhibitors for proliferating carnosine\nafter ingestion. Thus, molecular dynamics (MD) simulations were performed\nto explore the binding mechanism of BES to both CN1 and CN2. The binding\nof BES-CN1 and BES-CN2 was studied in comparison. The results indicated\nthat BES could bind both CNs with different degrees of binding affinity.\nBES prefers CN2 because: (1) its aryl terminus is trapped by Y197\nin an S1 pocket; (ii) the BES polar backbone is firmly bound by catalytic\nMn<sup>2+</sup> ions; and (iii) the S1′ pocket can shrink to\naccommodate the isopropyl end of BES. In contrast, the high mobility\nof the aryl end and the complete loss of metal–BES interactions\nin CN1 cause a loose BES binding. Seemingly, polar termini were required\nfor a good CN1 inhibitor.