Catalytic properties of thimet oligopeptidase H600A mutant

Catalytic properties of thimet oligopeptidase H600A mutant

Autor Machado, Mauricio F. M. Autor UNIFESP Google Scholar
Marcondes, Marcelo F. Autor UNIFESP Google Scholar
Rioli, Vanessa Google Scholar
Ferro, Emer S. Google Scholar
Juliano, Maria A. Autor UNIFESP Google Scholar
Juliano, Luiz Autor UNIFESP Google Scholar
Oliveira, Vitor Autor UNIFESP Google Scholar
Instituição Universidade Federal de São Paulo (UNIFESP)
Inst Butantan
Universidade de São Paulo (USP)
Resumo Thimet oligopeptidase (EC, TOP) is a metallo-oligopeptidase that participates in the intracellular metabolism of peptides. Predictions based on structurally analogous peptidases (Dcp and ACE-2) show that TOP can present a hinge-bend movement during substrate hydrolysis, what brings some residues closer to the substrate. One of these residues that in TOP crystallographic structure are far from the catalytic residues, but, moves toward the substrate considering this possible structural reorganization is His(600). in the present work, the role of His(600) of TOP was investigated by site-directed mutagenesis. TOP H600A mutant was characterized through analysis of S(1) and S(1)', specificity, pH-activity profile and inhibition by JA-2. Results showed that TOP His(600) residue makes important interactions with the substrate, supporting the prediction that His(600) moves toward the substrate due to a hinge movement similar to the Dcp and ACE-2. Furthermore, the mutation H600A affected both K(m) and k(cat), showing the importance of His(600) for both substrate binding and/or product release from active site. Changes in the pH-profile may indicate also the participation of His(600) in TOP catalysis, transferring a proton to the newly generated NH(2)-terminus or helping Tyr(605) and/or Tyr(612) in the intermediate oxyanion stabilization. (C) 2010 Elsevier Inc. All rights reserved.
Palavra-chave EP24.15
Substrate and inhibitor specificity
Site-directed mutagenesis
Thimet oligopeptidase
Idioma Inglês
Financiador Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Número do financiamento FAPESP: 08/57336-2
Data de publicação 2010-04-02
Publicado em Biochemical and Biophysical Research Communications. San Diego: Academic Press Inc Elsevier Science, v. 394, n. 2, p. 429-433, 2010.
ISSN 0006-291X (Sherpa/Romeo, fator de impacto)
Publicador Elsevier B.V.
Extensão 429-433
Direito de acesso Acesso restrito
Tipo Artigo
Web of Science WOS:000276732400033
Endereço permanente

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