A comparative scrutiny of various glucagon variants in our prior experimental study facilitated the localization of the catalytic activity to an enzyme-like pocket formed on the surface of the glucagon fibril, which includes the histidyl-serine domain at the peptide's N-terminus. Based on this knowledge, we carried out extensive docking and binding energy calculation of the different poses of the substrate p-NPA bound around this catalytic-dyad. This was followed by investigating into the catalytic mechanism using the EVB and PDLD/S-LRA-2000 methods.