Activity-Guided Discovery and Functional Characterization of VTPPHVE, a Tyrosinase-Inhibitory Peptide from Golden Pompano (Trachinotus anak) Muscle Proteins

Marine-derived bioactive peptides are promising natural enzyme inhibitors, but their functional robustness and inhibition behavior require experimental validation. Golden pompano (Trachinotus anak) muscle proteins were subjected to ultrasound-assisted neutral protease–papain hydrolysis, activity-guided ultrafiltration, LC–MS/MS identification, and synthetic-peptide validation. The <3 kDa ultrafiltration fraction showed the highest activity (75.43% tyrosinase inhibition and 51.40% apparent Cu2+ chelation) and was selected for LC–MS/MS. Five candidate peptides were synthesized after stepwise prioritization. VTPPHVE showed the lowest IC50 value for tyrosinase inhibition (113.41 μM) and exhibited the strongest apparent Cu2+-chelating activity among the synthesized peptides (141.54 μM). VTPPHVE retained comparable inhibitory activity after pH 2–10 treatment and heating at 20–100 °C, and showed detectable apparent activity after simulated gastrointestinal treatments, although the digested samples were not normalized to an equivalent peptide concentration at the assay stage and digestion-fluid blanks were not included. Enzyme kinetics indicated mixed-type inhibition, with a concentration-dependent decrease in apparent Vmax and an increase in apparent Km. Molecular docking placed VTPPHVE in an inter-chain surface region spanning H-subunit chains A and B, while a 100 ns molecular dynamics simulation supported the dynamic persistence of the docking-derived model after initial conformational adjustment. These results identify VTPPHVE as a marine fish-derived tyrosinase-inhibitory peptide and extend its characterization from sequence-level discovery to functional activity retention, enzyme kinetics, and exploratory structural modeling.