Effects of Mutations in the Carboxyl-Terminal Region on the Catalytic Activity of Escherichia coli Signal Peptidase I

Escherichia coli signal peptidase I (SPase I) is a membrane-bound serine endopeptidase that catalyses the cleavage of signal peptides from the pre-forms of membrane or secretory proteins. Our previous studies using chemical modification and site-directed mutagenesis suggested that Trp300 and Arg77,...

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Bibliographic Details
Published in:	Journal of biochemistry (Tokyo) 2008-02, Vol.143 (2), p.237-242
Main Authors:	Kim, Yong-Tae, Yoshida, Hisashi, Kojima, Masaki, Kurita, Ryo, Nishii, Wataru, Muramatsu, Tomonari, Ito, Hisashi, Park, Sun Joo, Takahashi, Kenji
Format:	Article
Language:	eng
Subjects:	Binding Sites carboxyl-terminal residues Catalysis deletion Electrophoresis, Polyacrylamide Gel Escherichia coli Escherichia coli - enzymology Kinetics Membrane Proteins - chemistry Membrane Proteins - genetics Membrane Proteins - metabolism Models, Molecular Mutagenesis Protein Conformation Serine Endopeptidases - chemistry Serine Endopeptidases - genetics Serine Endopeptidases - metabolism signal peptidase I site-directed mutagenesis
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Summary:	Escherichia coli signal peptidase I (SPase I) is a membrane-bound serine endopeptidase that catalyses the cleavage of signal peptides from the pre-forms of membrane or secretory proteins. Our previous studies using chemical modification and site-directed mutagenesis suggested that Trp300 and Arg77, Arg222, Arg315 and Arg318 are important for the proper and stable conformation of the active site of SPase I. Interestingly, many of these residues reside in the C-terminal region of the enzyme. As a continuation of these studies, we investigated in the present study the effects of mutations in the C-terminal region including amino acid residues at positions from 319 to 323 by deletions and site-directed mutagenesis. As a result, the deletion of the C-terminal His323 was shown to scarcely affect the enzyme activity of SPase I, whereas the deletion of Gly321-His323 or Ile319-His323 as well as the point mutation of Ile322 to alanine was shown to decrease significantly both the activity in vitro and in vivo without a big gross conformational change in the enzyme. These results suggest a significant contribution of Ile322 to the construction and maintenance of the proper and critical local conformation backing up the active site of SPase I.
ISSN:	0021-924X 1756-2651