Screening and mechanistic investigation of food-derived multifunctional ACE-inhibitory tri-peptides

Food-derived angiotensin-converting enzyme (ACE)-inhibitory peptides (ACEIPs) with antioxidant and antibacterial properties can function as food additives to alleviate hypertension-induced oxidative stress and bacterial infections. This study used bioinformatics to analyze Gly-Xaa-Pro ACEIPs, identifying Gly-Pro-Pro (GPP) from buckwheat. GPP exhibited potent free-radical-scavenging capacity and at 4 mg/mL eradicated both E. coli and S. aureus (0 % survival vs 14.1 %/4.6 % with GRP; p < 0.001), demonstrating superior antioxidant and bactericidal potency. Multispectral techniques, thermodynamic analysis, and molecular docking simulations were used for a detailed molecular-level investigation to better understand the binding mechanism between GPP and ACE. The results indicated that GPP's binding to ACE is non-competitive and acts as an inhibitor. The inhibitory effect of GPP on ACE from its moderate binding ( K a =1.525×10 4 M −1 ), which alters the microenvironment and conformation of ACE. The binding process is primarily driven by entropic forces arising from hydrophobic interactions and proceeds spontaneously due to ΔG<0. Through multi-molecular synergy (Hill coefficient n =513), GPP forms hydrogen bonds with the Asp 121, Glu 123, Tyr 135, and Trp 220 residue sites outside ACE's active center, thereby inhibiting ACE activity. In conclusion, the research of GPP provides a theoretical basis for developing multifunctional blood-pressure-lowering dietary supplements. • Buckwheat-derived Gly-Pro-Pro (GPP) was screened via in silico and experiments. • GPP is an ACE-inhibitory peptide with antioxidant and antimicrobial properties. • GPP exhibited non-competitive inhibition against ACE. • Multiple GPPs bound to ACE and induced conformational changes. • GPP-ACE complex was stabilized through multiple non-covalent forces.