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.