Why Bestatin Prefers Human Carnosinase 2 (CN2) to Human Carnosinase 1 (CN1).

Abstract

Human carnosinases (CNs) are Xaa-His metal-ion-activated aminopeptidases that break down bioactive carnosine and other histidine-containing dipeptides. Carnosine is a bioactive peptide found in meat and prevalently used as a supplement and in functional food formulation. Nonetheless, carnosine is digested by CNs rapidly after ingestion. CNs have two isoforms (carnosinase 1 (CN1) and carnosinase 2 (CN2)), where CN1 is the main player in carnosine digestion. CNs contain a catalytic metal ion pair (Zn²⁺ for CN1 and Mn²⁺ for CN2) and two subpockets (S1 and S1' pockets) to accommodate a substrate. Bestatin (BES) has been reported to be active for CN2; however, its inhibition ability for CN1 has remained under debate, because the underlying mechanism remains unclear. This information is important for designing novel CN1-selective inhibitors for proliferating carnosine after ingestion. Thus, molecular dynamics (MD) simulations were performed to explore the binding mechanism of BES to both CN1 and CN2. The binding of BES-CN1 and BES-CN2 was studied in comparison. The results indicated that BES could bind both CNs with different degrees of binding affinity. BES prefers CN2 because: (1) its aryl terminus is trapped by Y197 in an S1 pocket; (ii) the BES polar backbone is firmly bound by catalytic Mn²⁺ ions; and (iii) the S1' pocket can shrink to accommodate the isopropyl end of BES. In contrast, the high mobility of the aryl end and the complete loss of metal-BES interactions in CN1 cause a loose BES binding. Seemingly, polar termini were required for a good CN1 inhibitor.