Salt Effect on Substrate Specificity of a Subtilisin-Like Halophilic Protease

Salt Effect on Substrate Specificity of a Subtilisin-Like Halophilic Protease

Author Okamoto, Débora Noma Autor UNIFESP Google Scholar
Kondo, Marcia Yuri Autor UNIFESP Google Scholar
Hiraga, Kazumi Google Scholar
Juliano, Maria Aparecida Autor UNIFESP Google Scholar
Juliano, Luiz Autor UNIFESP Google Scholar
Gouvea, Iuri Estrada Autor UNIFESP Google Scholar
Oda, Kohei Google Scholar
Institution Universidade Federal de São Paulo (UNIFESP)
Kyoto Inst Technol
Abstract Enzyme-substrate interaction under the presence of high concentration of salts is of great interest for biotechnology applications and basic enzymology. In our previous work, the salt effect on halophilic subtilase SR5-3 was evaluated with Suc-AAPF-MCA and with the FRET peptide Abz-AAPFSSKQ-EDDnp. It was demonstrated that the magnitude of catalytic activity enhancement was affected by the presence of the prime site residues. In this work, a detailed analysis of the salt effect on SR5-3 protease substrate specificity was performed using chromogenic and coumarin substrates as well as FRET peptides derived from Abz-KLRSSKQ-EDDnp. The followings were demonstrated: 1) Preference of amino acid of SR5-3 protease at the P(3), P(2), P(1), P(1)' or P(2)' position of FRET substrates was almost similar with that of subtilisin. 2) Under the presence of the salts (3M NaCl or 1M Na(2)SO(4)), SR5-3 protease showed higher kcat values, lower Km values and totally 2-6 times higher kcat/Km values compared with those of control for FRET substrates, and salts did not significantly affect the preference of amino acid residues at the primary positions (P1 and P1'), but it affected the preference at the P(2) and P(2)' position. In contrast, for smaller substrates with only non-prime sites, SR5-3 protease showed 20-75 times higher kcat/Km values compared with those of control. These findings are in agreement with the notion that increases in enzyme-substrate interactions in subtilases alter the rate-determining step in peptide hydrolysis.
Keywords Peptidase
FRET peptides
kosmotropic salts
Language English
Sponsor Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Date 2010-06-01
Published in Protein And Peptide Letters. Sharjah: Bentham Science Publ Ltd, v. 17, n. 6, p. 796-802, 2010.
ISSN 0929-8665 (Sherpa/Romeo, impact factor)
Publisher Bentham Science Publ Ltd
Extent 796-802
Origin http://dx.doi.org/10.2174/092986610791190363
Access rights Closed access
Type Article
Web of Science ID WOS:000277143400016
URI http://repositorio.unifesp.br/11600/45144

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