Enzymolysis Modes Trigger Diversity in Inhibitor-α-Amylase Aggregating Behaviors and Activity Inhibition: A New Insight Into Enzyme Inhibition.

Inhibitors of α-amylase have been developed to regulate postprandial blood glucose fluctuation. The enzyme inhibition arises from direct or indirect inhibitor-enzyme interactions, depending on inhibitor structures. However, an ignored factor, substrate, may also influence or even decide the enzyme inhibition. In this work, it is innovatively found that the difference in substrate enzymolysis modes, i.e., structural composition and concentration of α-1,4-glucosidic bonds, triggers the diversity in inhibitor-enzyme aggregating behaviors and α-amylase inhibition. For competitive inhibition, there exists an equilibrium between α-amylase-substrate catalytic affinity and inhibitor-α-amylase binding affinity; therefore, a higher enzymolysis affinity and concentration of α-1,4-glucosidic structures interferes the balance, unfavoring inhibitor-enzyme aggregate formation and thus weakening α-amylase inhibition. For uncompetitive inhibition, the presence of macromolecular starch is necessary instead of micromolecular GalG2CNP, which not only binds with active site but with an assistant flexible loop (involving Gly(304)-Gly(309)) near the site. Hence, the refined enzyme structure due to the molecular flexibility more likely favors the inhibitor binding with the non-active loop, forming an inhibitor-enzyme-starch ternary aggregate. Conclusively, this study provides a novel insight into the evaluation of α-amylase inhibition regarding the participating role of substrate in inhibitor-enzyme aggregating interactions, emphasizing the selection of appropriate substrates in the development and screening of α-amylase inhibitors.