Structure-activity relationship and molecular interaction mechanisms of the ACE-inhibitory tripeptide LL-X.

Investigating the molecular interaction mechanisms of angiotensin-converting enzyme (ACE) inhibitory peptides is valuable in antihypertensive drug development. In this study, we designed two novel peptide variants, Leu-Leu-Phe (LLF) and Leu-Leu-Pro (LLP), by substituting the C-terminal tyrosine (Tyr) in casein-derived Leu-Leu-Tyr (LLY) with phenylalanine (Phe) and proline (Pro), respectively, to investigate the impact of C-terminal amino acid properties of peptide on ACE inhibition. The ACE inhibitory activity, inhibition kinetics, peptides-ACE binding mechanisms, and LLF and LLP conformational relationship were systematically investigated with comparative assessment against the LLY. The results showed the ACE inhibitory activity of the synthetic peptide (LLF: IC50 = 168.57 ± 5.11 μM; LLP: IC50 = 96.64 ± 2.93 μM) was significantly lower than that casein-derived tripeptide LLY (IC50 = 44.16 ± 2.45 μM). The Lineweaver-Burk plots and molecular docking indicated that both LLF and LLP were noncompetitive inhibitors, which was consistent with LLY. Molecular dynamics simulations validated their stable binding to the ACE complex. The comparison of the three peptides interacting with ACE using multispectral techniques indicated that LLY resulted in the formation of a greater number of disordered protein structural regions, rendering it more active than the other peptides. Consequently, we concluded that the role of the C-terminal Tyr in the ACE inhibitory peptide LLY is more significant than that of Phe and Pro. This study highlights the crucial influence of C-terminal amino acids on the inhibitory activity of tripeptides, providing an essential theoretical foundation for the rational design of effective ACE inhibitory peptides.