Tk-subtilisin from the hyperthermophilic archaeon Thermococcus kodakarensis matures from Pro-Tk-subtilisin (Pro-TKS) upon autoprocessing and degradation of propeptide. Pro-TKS contains the insertion sequence (IS1) at the N-terminus of the mature domain as compared to bacterial pro-subtilisins. To analyze the role of IS1, the Pro-TKS derivative without IS1 (∆IS1-Pro-TKS) and its active-site mutants (∆IS1-Pro-S324A and ∆IS1-Pro-S324C) were constructed and characterized. ∆IS1-Pro-S324A and ∆IS1-Pro-TKS represent an unautoprocessed and autoprocessed form of ∆IS1-Pro-TKS, respectively. The CD and ANS fluorescence spectra of these proteins indicate that folding of ∆IS1-Pro-TKS is not completed by binding of Ca(2+) ions but is completed by the subsequent autoprocessing reaction. Thermal denaturation of these proteins analyzed by DSC and CD spectroscopy indicates that unautoprocessed ∆IS1-Pro-TKS is less stable than autoprocessed ∆IS1-Pro-TKS by 26.3 °C in T (m). The stability of autoprocessed ∆IS1-Pro-TKS is comparable to that of Pro-TKS, which is slightly lower than that of unautoprocessed Pro-TKS. These results suggest that ∆IS1-Pro-TKS is fully folded and greatly stabilized by autoprocessing. ∆IS1-Pro-TKS more slowly matured to ∆IS1-Tk-subtilisin than Pro-TKS did, due to a decrease in the autoprocessing rate. We propose that IS1 is required not only for hyperstabilization of Pro-TKS but also for its rapid maturation.