Asp72 of Pro-Peptide is an Important pH Sensor in the Zymogen Activation Process of Papain:A Structural and Mechanistic Insight

The zymogen of papain contains a pro-peptide at the N-terminus of the catalytic domain. Pro-peptide contains residues which act as pH-sensors in zymogen activation cascade. To understand the influence of pro-peptide in the pH-induced zymogen activation process of the protease, we performed structural studies of the zymogen of papain at activation pH of 4.0. The X-ray structure of zymogen at acidic pH reveals that Asp72 of the pro-peptide, a highly conserved residue of the GXNXFXD motif, plays an important role in pH-induced conformational changes in the prodomain leading to the zymogen activation process. Far-UV circular dichroism spectrum of zymogen at pH 4.0 demonstrates loss of helical structure compared to that at pH 8.0. The structural observation is further corroborated by mutational studies, where D72A mutant is shown to undergo auto-activation at pH 5.0 compared to pH 4.0 for wild-type, though the general proteolytic activity of the D72A mutant remains similar to that of wild-type. Our findings indicate that the conserved Asp72 residue is an important pH sensor in the zymogen activation process and that the pro-peptide part can also be a useful target of protein engineering for altering the activation pH of a protease.