Specific Substrate Activity of Lotus Root Polyphenol Oxidase: Insights from Gaussian-Accelerated Molecular Dynamics and Markov State Models

Polyphenol oxidase (PPO) plays a key role in the enzymatic browning process, and this study employed Gaussian-accelerated molecular dynamics (GaMD) simulations to investigate the catalytic efficiency mechanisms of lotus root PPO with different substrates, including catechin, epicatechin, and chlorogenic acid, as well as the inhibitor oxalic acid. Key findings reveal significant conformational changes in PPO that correlate with its enzymatic activity. Upon substrate binding, the alpha-helix in the Q53-D63 region near the copper ion extends, likely stabilizing the active site and enhancing catalysis. In contrast, this helix is disrupted in the presence of the inhibitor, resulting in a decrease in enzymatic efficiency. Additionally, the F350-V378 region, which covers the substrate-binding site, forms an alpha-helix upon substrate binding, further stabilizing the substrate and promoting catalytic function. However, this alpha-helix does not form when the inhibitor is bound, destabilizing the binding site and contributing to inhibition. These findings offer new insights into the substrate-specific and inhibitor-induced structural dynamics of lotus root PPO, providing valuable information for enhancing food processing and preservation techniques.