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An alternative mature form of subtilisin homologue, Tk‐SP, from Thermococcus kodakaraensis identified in the presence of Ca 2+
Author(s) -
Sinsereekul Nitat,
Foophow Tita,
Yamanouchi Mai,
Koga Yuichi,
Takano Kazufumi,
Kanaya Shigenori
Publication year - 2011
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2011.08107.x
Subject(s) - protein precursor , thermococcus , subtilisin , chemistry , biochemistry , microbiology and biotechnology , biology , enzyme , gene , archaea
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.