Many proteases require the assistance of an intramolecular chaperone (IMC) that is essential for protein folding. Subtilisin is produced as a precursor that requires its N-terminal propeptide to act as an IMC to chaperone the folding of its subtilisin domain. During the precursor folding, the cleavage of the peptide bond between the IMC and the subtilisin domain is the most important and rate-limiting step, which leads to the structural reorganization of the subtilisin domain and IMC's degradation. It is speculated that the cleavage is fulfilled by the nucleophilic attack of Ser221, with the assistance of Asp32 positioning the correct tautomer of His64 and His64 accepting a proton from Ser221. In this study, our results suggested that there was a different mechanism of cleavage of the peptide bond between the IMC and the subtilisin domain in nattokinase (NK), and the role of the NK catalytic triad on the cleavage was not consistent with the classical theory. This finding suggested that members of the subtilisin family had evolved different mechanisms to acquire their own active subtilisin efficiently.
Keywords: NK; catalytic triad; cleavage; intramolecular chaperone.