Characterization of blueberry (Vaccinium corymbosum L.) catechol oxidases III binding mechanism in response to selected substrates and inhibitors

Abstract

Catechol oxidase (CO) is one of the enzymes that cause browning of blueberries and related products. In-depth study of its properties will help reduce the loss caused by it. The optimal pH for catechol (CAT), 3,4-dihydroxyphenylacetic acid (DOPAC), 4-methylcatechol (4-MeCAT), 3-hydroxytyramine hydrochloride (3-HTH), and pyrogallol (PG) substrates were 6.0, 4.0, 3.0, 6.5, and 4.5, respectively. The substrates with catechol structure selected in the experiment can react with CO, except protocatechuic acid (PCA). Caffeic acid (CA) may be the most suitable natural substrate for this blueberry CO. The K⁺ and Na⁺ have little effect on the CO activity. Li⁺, Mg²⁺, Cu²⁺, Zn²⁺, and Ca²⁺ could increase enzyme activity at low concentrations (0–5 mmol/L). The most effective inhibitor in the experiment was tropolone (TPL; IC₅₀ = 10.01 ± 0.11 μmol/L), followed by 1,4-benzoquinone (1,4-BQ; IC₅₀ = 34.84 ± 0.56 μmol/L). It was found that the carboxyl or phenolic hydroxyl groups in the substrates and inhibitors played an important role in binding to the catalytic cavity. The position of THR320 (H_B1 + 1) was a key in regulating enzyme activity. The sugars should be in high concentration condition to inhibit enzyme activity.