Aminopeptidase and carboxypeptidase activity of DPP-4 on the example of peptides LPQNIPPL and LPβ³hQNIPPL

Our previous research demonstrated that LPQNIPPL and LPβ³hQNIPPL peptides are degraded in vitro by DPP-4 enzyme. Besides the expected degradation products, additional degradation products were observed on HPLC-MS chromatograms, suggesting cleavage of both the C- and N-terminal amino acids. Molecular docking provided a model for the observed carboxypeptidase, but not for aminopeptidase activity. Compared to conventional N-terminal binding at the active site, the obtained carboxypeptidase model suggested the α-amino group interaction with S2 subsite, C-terminal amino acid side chain interaction with the S1 subsite, reversed peptide backbone order and P1' proline in a secondary peptide bond. The scissile bond position and distance relative to the active Ser630 seems to remain similar compared to conventional binding. Efficient C-terminal binding to the DPP-4 active site is reinforced by molecular dynamics results, but with altered interactions compared to molecular docking after ligand adaptation phase. Although peptide interactions and distance of scissile bond from the DPP-4 nucleophile Ser630 appears to support the cleavage model, the required nucleophilic attack angles are not confirmed. The proposed model does not allow final conclusions on nucleophilic attack stereochemistry but opens the discussion on possible DPP-4 carboxypeptidase cleavage of peptides, as well as retro/retro-inverso peptides with C-terminal Leu-NH₂.