title: 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.
abstract: 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.
authors.affiliation: Department of Material and Life Science, Graduate School of Engineering, Osaka University, Suita, Osaka, 565-0871, Japan.
authors.forename: Kota
authors.initials: K
authors.lastname: Yuzaki
authors.forename: Yudai
authors.initials: Y
authors.lastname: Sanda
authors.forename: Dong-Ju
authors.initials: DJ
authors.lastname: You
authors.forename: Ryo
authors.initials: R
authors.lastname: Uehara
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Koga
authors.forename: Shigenori
authors.initials: S
authors.lastname: Kanaya
mesh_terms: D019943:Amino Acid Substitution; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D058849:Protein Refolding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D020860:Subtilisin / Q000737:chemistry* / Q000235:genetics; D013381:Subtilisins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019712:Thermococcus / Q000235:genetics / Q000378:metabolism
chemical_list: D019843:Archaeal Proteins; D004792:Enzyme Precursors; D010446:Peptide Fragments; C057107:prosubtilisin; D013381:Subtilisins; D020860:Subtilisin
keywords: Thermococcus kodakarensis; activation, hydrophobic core; propeptide; subtilisin