Elucidating the mechanism of amylolytic enzyme activity inhibition by rice glutelin amyloid fibril aggregates

The inhibitory mechanisms of rice glutelin amyloid fibril aggregates (RAFA) against amylolytic enzymes (α-amylase and amyloglucosidase) were examined by enzyme kinetic analysis, fluorescence quenching, and molecular dynamics (MD) simulations. The results of kinetic studies revealed that 2 h-RAFA exhibited anticompetitive inhibition against α-amylase, while both 6 h-RAFA and 10 h-RAFA demonstrated competitive inhibition. For amyloglucosidase, different RAFA both exhibited competitive inhibition. In fluorescence spectroscopy analysis, RAFA led to a decrease in fluorescence intensity and a red-shift in the maximum emission wavelengths of amylolytic enzymes, suggesting evident interactions between RAFA and the enzymes. Furthermore, the MD simulations further indicated that RAFA bound to specific amino acid residues near the active site of α-amylase through hydrogen bonding, electrostatic interactions, and hydrophobic interactions. This interaction resulted in a modified microenvironment surrounding the Tyr residue in the enzyme's structure, ultimately leading to inhibited enzyme activity. In the case of amyloglucosidase, RAFA was observed to bind primarily to the catalytic structural domain of the enzyme via hydrogen bonding and hydrophobic interactions, effectively blocking the substrate's access and, consequently, diminishing enzyme activity. In summary, this study establishes a theoretical foundation for understanding the mechanisms by which RAFA inhibits starch digestion.