Please use this identifier to cite or link to this item: https://repositorio.unifesp.br/handle/11600/32466
Title: Catalytic properties of thimet oligopeptidase H600A mutant
Authors: Machado, Mauricio F. M. [UNIFESP]
Marcondes, Marcelo F. [UNIFESP]
Rioli, Vanessa
Ferro, Emer S.
Juliano, Maria A. [UNIFESP]
Juliano, Luiz [UNIFESP]
Oliveira, Vitor [UNIFESP]
Universidade Federal de São Paulo (UNIFESP)
Inst Butantan
Universidade de São Paulo (USP)
Keywords: EP24.15
Substrate and inhibitor specificity
Site-directed mutagenesis
Thimet oligopeptidase
Issue Date: 2-Apr-2010
Publisher: Elsevier B.V.
Citation: Biochemical and Biophysical Research Communications. San Diego: Academic Press Inc Elsevier Science, v. 394, n. 2, p. 429-433, 2010.
Abstract: Thimet oligopeptidase (EC 3.4.24.15, 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.
URI: http://repositorio.unifesp.br/handle/11600/32466
ISSN: 0006-291X
Other Identifiers: http://dx.doi.org/10.1016/j.bbrc.2010.03.045
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