In summary, our study involved a comprehensive exploration of the acylation step in the p-NPA esterolysis process, catalyzed by glucagon amyloid fibrils. By employing the stateof-the-art EVB method and PDLD/S-LRA-2000 approach, we unraveled the intricacies of this reaction mechanism, treating it as an instructive test for the catalytic capabilities of glucagon amyloid fibrils-a topic we had previously investigated through experimental means.