Isolation, Purification, Identification, and In Vitro ACE Inhibitory Activity of Tyrosine‐Arginine Dipeptide From Red Millet Huangjiu

This study aims to isolate and identify endogenous peptides with angiotensin‐converting enzyme (ACE) inhibitory activity from red millet Huangjiu and to systematically elucidate their molecular mechanisms of action. Utilizing ultrafiltration, gel chromatography, and reversed‐phase high‐performance liquid chromatography (RP‐HPLC) followed by electrospray ionization tandem mass spectrometry (ESI‐MS/MS) analysis, the tyrosine‐arginine dipeptide (Tyr‐Arg) was successfully identified. In vitro assays demonstrated that Tyr‐Arg possesses moderate ACE inhibitory activity, with a half‐maximal inhibitory concentration (IC 50 ) value of 158.93 ± 0.39 μM. Moreover, molecular docking analysis revealed that Tyr‐Arg binds to ACE via hydrogen bonding, π‐alkyl interactions, and van der Waals forces. Notably, the binding site was located outside the catalytic core, suggesting that Tyr‐Arg functions as a non‐competitive or allosteric inhibitor by inducing conformational changes in the enzyme. This mechanism was further corroborated by molecular dynamics (MD) simulations, which showed that the binding triggers a transition to a more open enzyme state, thereby obstructing substrate recognition or product release. MD simulations, integrated with molecular mechanics/generalized born surface area (MM/GBSA) calculations, substantiated that the Tyr‐Arg/ACE complex underwent significant structural expansion, as evidenced by the increased radius of gyration ( Rg ) and solvent‐accessible surface area (SASA), which stabilized after 80 ns in the late stage of the simulation. Despite the extensive expansion and the consequent high root mean square deviation (RMSD), the complex achieved a stable binding conformation during the final 20 ns, yielding a total binding free energy of −7.88 kcal/mol. The binding mechanism is primarily driven by a synergy of non‐polar interactions and electrostatic attraction; however, the electrostatic component is notably offset by the energetic costs associated with polar desolvation. Additionally, free energy landscape (FEL) analysis revealed the step‐wise sequential conformational transition pathway, identifying the global energy minima that stabilize the complex and prevent ligand dissociation, even in the face of substantial structural rearrangements observed during the 100 ns simulation. This study elucidates the molecular basis of ACE inhibition by Tyr‐Arg, providing a scientific foundation and novel insights for the valorization of functional components in traditional fermented foods.