Increase in activation rate of Pro‐Tk‐subtilisin by a single nonpolar‐to‐polar amino acid substitution at the hydrophobic core of the propeptide domain

Tk‐subtilisin (Gly70‐Gly398) is a subtilisin homolog from Thermococcus kodakarensis. Active Tk‐subtilisin is produced from its inactive precursor, Pro‐Tk‐subtilisin (Gly1‐Gly398), by autoprocessing and degradation of the propeptide (Tk‐propeptide, Gly1‐Leu69). This activation process is extremely slow at moderate temperatures owing to high stability of Tk‐propeptide. Tk‐propeptide is stabilized by the hydrophobic core. To examine whether a single nonpolar‐to‐polar amino acid substitution at this core affects the activation rate of Pro‐Tk‐subtilisin, the Pro‐Tk‐subtilisin derivative with the Phe17→His mutation (Pro‐F17H), Tk‐propeptide derivative with the same mutation (F17H‐propeptide), and two active‐site mutants of Pro‐F17H (Pro‐F17H/S324A and Pro‐F17H/S324C) were constructed. The crystal structure of Pro‐F17H/S324A was nearly identical to that of Pro‐S324A, indicating that the mutation does not affect the structure of Pro‐Tk‐subtilisin. The refolding rate of Pro‐F17H/S324A and autoprocessing rate of Pro‐F17H/S324C were also nearly identical to those of their parent proteins (Pro‐S324A and Pro‐S324C). However, the activation rate of Pro‐F17H greatly increased when compared with that of Pro‐Tk‐subtilisin, such that Pro‐F17H is efficiently activated even at 40°C. The far‐UV circular dichroism spectrum of F17H‐propeptide did not exhibit a broad trough at 205–230 nm, which is observed in the spectrum of Tk‐propeptide. F17H‐propeptide is more susceptible to chymotryptic degradation than Tk‐propeptide. These results suggest that F17H‐propeptide is unfolded in an isolated form and is therefore rapidly degraded by Tk‐subtilisin. Thus, destabilization of the hydrophobic core of Tk‐propeptide by a nonpolar‐to‐polar amino acid substitution is an effective way to increase the activation rate of Pro‐Tk‐subtilisin.