title: An alternative mature form of subtilisin homologue, Tk-SP, from Thermococcus kodakaraensis identified in the presence of Ca2+.
abstract: Pro-Tk-SP from Thermococcus kodakaraensis consists of the four domains: N-propeptide, subtilisin (EC 3.4.21.62) domain, β-jelly roll domain and C-propeptide. To analyze the maturation process of this protein, the Pro-Tk-SP derivative with the mutation of the active-site serine residue to Cys (Pro-Tk-S359C), Pro-Tk-S359C derivatives lacking the N-propeptide (ProC-Tk-S359C) and both propeptides (Tk-S359C), and a His-tagged form of the isolated C-propeptide (ProC*) were constructed. Pro-Tk-S359C was purified mostly in an autoprocessed form in which the N-propeptide is autoprocessed but the isolated N-propeptide (ProN) forms a stable complex with ProC-Tk-S359C, indicating that the N-propeptide is autoprocessed first. The subsequent maturation process was analyzed using ProC-Tk-S359C, instead of the ProN:ProC-Tk-S359C complex. The C-propeptide was autoprocessed and degraded when ProC-Tk-S359C was incubated at 80 °C in the absence of Ca(2+). However, it was not autoprocessed in the presence of Ca(2+). Comparison of the susceptibility of ProC* to proteolytic degradation in the presence and absence of Ca(2+) suggests that the C-propeptide becomes highly resistant to proteolytic degradation in the presence of Ca(2+). We propose that Pro-Tk-SP derivative lacking N-propeptide (Val114-Gly640) represents a mature form of Pro-Tk-SP in a natural environment. The enzymatic activity of ProC-Tk-S359C was higher than (but comparable to) that of Tk-S359C, suggesting that the C-propeptide is not important for activity. However, the T(m) value of ProC-Tk-S359C determined by far-UV CD spectroscopy was higher than that of Tk-S359C by 25.9 °C in the absence of Ca(2+) and 7.5 °C in the presence of Ca(2+), indicating that the C-propeptide contributes to the stabilization of ProC-Tk-S359C.
authors: Department of Material and Life Science, Graduate School of Engineering, Osaka University, Japan.
authors: Nitat
authors: N
authors: Sinsereekul
authors: Tita
authors: T
authors: Foophow
authors: Mai
authors: M
authors: Yamanouchi
authors: Yuichi
authors: Y
authors: Koga
authors: Kazufumi
authors: K
authors: Takano
authors: Shigenori
authors: S
authors: Kanaya
mesh_terms: D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D002942:Circular Dichroism; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D008958:Models, Molecular; D008970:Molecular Weight; D009154:Mutation; D011499:Protein Processing, Post-Translational; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020860:Subtilisin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019712:Thermococcus / Q000201:enzymology*
chemical_list: D017931:DNA Primers; D020860:Subtilisin; D002118:Calcium