Studies of Inhibitory Effects of p-Coumaric Acid, Kojic Acid and Chlorogenic Acid on Different Forms of Polyphenol Oxidase from Apple

Apple often contains two forms of polyphenol oxidase (PPO), including membrane-bound polyphenol oxidase (mPPO) and soluble polyphenol oxidase (sPPO). These two forms differ in their enzymatic properties and structural characteristics. In this study, sPPO and mPPO were isolated and purified from apples to evaluate their optimal pH and temperature, and investigate the inhibitory effects of p-coumaric acid, kojic acid, and chlorogenic acid. The results showed that the molecular weight, optimal pH and optimal temperature of sPPO were 30 kDa, 7.0, and 45 ℃, respectively, whereas those of mPPO were 67 kDa, 7.5, and 55 ℃. Structural analysis using amino acid sequencing and homology modeling revealed that sPPO primarily consisted of six α-helices, two β-folds and multiple random coils, while mPPO was mainly composed of eight α-helices, three β-folds and multiple random coils. Regarding inhibitory effects, p-coumaric acid and kojic acid were more effective in inhibiting PPO than chlorogenic acid, with sPPO being more susceptible to inhibition by these two acids compared to mPPO. Furthermore, both sPPO and mPPO were reversibly inhibited by all three inhibitors. Specifically, p-coumaric acid and kojic acid acted as competitive inhibitors, where chlorogenic acid demonstrated mixed inhibition. All three inhibitors were found to bind to a single site on PPO, significantly reducing its fluorescence. Molecular docking results indicated that the inhibitors primarily interacted with PPO though hydrogen bonding and hydrophobic interaction. The inhibitors could access the double copper-binding region of sPPO, but in mPPO, binding was restricted to the hydrophobic cavity. Molecular dynamics simulations further showed that the sPPO system exhibited greater fluctuations and flexibility, as indicated by RMSD, RMSF and Rg values. The addition of inhibitors significantly reduced the stability of the sPPO system compared to the mPPO system. This study provides a valuable insight into the development of effective and green PPO inhibitors, and offers a theoretical foundation for future research into the mechanisms of PPO inhibition.