Inconsistency between polyphenol-enzyme binding interactions and enzyme inhibition: Galloyl moiety decreases amyloglucosidase inhibition of catechins

Inhibiting carbohydrate-hydrolyzing enzymes has been considered as an effective approach for controlling starch digestion and postprandial blood glucose level. α-Amylase and amyloglucosidase (AMG) are commonly applied in analysis of starch digestion behaviour. Catechins have been shown with the inhibiting effects on α-amylase. However, the inhibitory activity of catechins against AMG needs to be further studied. Therefore, AMG inhibition of 8 catechins and the mechanisms were studied in this work through substrate depletion, inhibition kinetics, molecular docking, fluorescence quenching, differential scanning calorimetry, and isothermal titration calorimetry. The inhibitory activity of catechins with galloyl moiety (CGMs) was found to be lower than the corresponding catechins without the moiety (Cs). All catechins were anti-competitive inhibitors, indicating that they tended to bind with AMG-starch complex in the digestion system, rather than with AMG directly. Interestingly, CGMs had higher quenching effects on AMG fluorescence than Cs, due to the additional π-stacking between aromatic rings of GM and AMG fluorophores. Also, CGMs had a higher binding affinity to AMG, due to the tendency of GM to AMG active site, although the affinity was much weaker than that of starch to AMG. Besides, catechins did not affect AMG thermostability. Therefore, there was an inconsistency between catechins-AMG binding interactions and the enzyme inhibition because the predominant sites for catechins binding were the non-active sites on AMG-starch complex, rather than the enzyme active ones. Conclusively, inhibition mode should also be considered when evaluating the inhibitory activity of a polyphenol based on the polyphenol-enzyme binding affinity.