A CLN2-Related and Thermostable Serine-Carboxyl Proteinase, Kumamolysin: Cloning, Expression, and Identification of Catalytic Serine Residue.
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概要
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The gene encoding kumamolysin, a thermostable pepstatin-insensitive carboxyl proteinase, was cloned and expressed. (i) Kumamolysin was synthesized as a large precursor consisting of two regions: amino-terminal prepro (188 amino acids) and mature proteins (384 amino acids). (ii) The deduced amino acid sequence of the mature region exhibited high similarity to those of such bacterial pepstatin-insensitive enzymes as Pseudomonas carboxyl proteinase (PSCP; EC 3.4.23.37, identity = 37%), Xanthomonas carboxyl proteinase (XCP; EC 3.4.23.33, identity = 36%), and human CLN2 gene product (identity = 36%), which is related to a fatal neurodegenerative disease. (iii) The presumed catalytic triad, Glu78, Asp82, Ser278 [three-dimensional structure of PSCP: Wlodawer, A. et al. (2001) Nature Struct. Biol., 8, 442-446], was found to be conserved in the amino acid sequence of kumamolysin. (iv) Kumamolysin was inactivated by such aldehyde-type inhibitors as Ac-Ile-Pro-Phe-CHO (Ki = 0.7 ± 0.14 μM). In PSCP, it has been clarified that these inhibitors form a hemiacetal linkage with the catalytic serine resi-due and inactivate the enzyme. (v) Mutational analysis of the Ser278 residue revealed that the mutant lost both auto-processing activity and proteolytic activity. These results strongly suggest that kumamolysin has a unique catalytic triad consisting of Glu78, Asp82, and Ser278 residues, as previously observed for PSCP.
- 社団法人 日本生化学会の論文
著者
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Oyama Hiroshi
Department Of Applied Biology Faculty Of Textile Science Kyoto Institute Of Technology
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BEYER Bret
Department of Biochemistry and Molecular Biology, University of Florida College of Medicine
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Oda Kohei
Department Of Agricultural Chemistry College Of Agriculture University Of Osaka Prefecture
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Dunn Ben
Department Of Biochemistry And Molecular Biology University Of Florida
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Beyer Bret
Department Of Biochemistry And Molecular Biology University Of Florida College Of Medicine
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Murao Sawao
Department Of Agricultural Chemistry College Of Agriculture University Of Osaka Prefecture
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Ogasawara Shin
Department Of Applied Biology Faculty Of Textile Science Kyoto Institute Of Technology
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Uchida Kenichi
Department Of Bioscience School Of Science And Engineering Teikyo University
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Hamada Takatoshi
Department Of Applied Microbial Technology Kumamoto Institute Of Technology
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