Tk-subtilisin (a subtilisin homologue from Thermococcus kodakaraensis) is matured from Pro-Tk-subtilisin upon autoprocessing and degradation of Tk-propeptide. To analyze the folding mechanism of Tk-subtilisin, the crystal structure of the active site mutant of Tk-subtilisin (S324A-subtilisin*), which was refolded in the presence of Ca2+ and absence of Tk-propeptide, was determined at 2.16A resolution. This structure is essentially the same as that of Tk-subtilisin matured from Pro-Tk-subtilisin. S324A-subtilisin* was refolded with a rate constant of 0.17 and 1.8min(-1) at 30 degrees C in the absence and presence of Tk-propeptide, respectively, indicating that Tk-subtilisin does not require Tk-propeptide for folding but requires it for acceleration of folding.