Catalytic properties of thimet oligopeptidase H600A mutant

Autor(es)

Machado, Mauricio F. M. ^[1] ; Marcondes, Marcelo F. ^[1] ; Rioli, Vanessa ^{[2, 3]} ; Ferro, Emer S. ^[2] ; Juliano, Maria A. ^[1] ; Juliano, Luiz ^[1] ; Oliveira, Vitor ^[1]

Afiliação do(s) autor(es)

^[1] Univ Fed Sao Paulo, Dept Biophys, BR-04044020 Sao Paulo - Brazil

^[2] Univ Sao Paulo, Dept Biol Celular & Desenvolvimento, BR-05508900 Sao Paulo - Brazil

^[3] Inst Butantan, Lab Especial Toxinol Aplicada, BR-05467010 Sao Paulo - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Biochemical and Biophysical Research Communications; v. 394, n. 2, p. 429-433, APR 2 2010.
• Citações Web of Science: 1

Resumo

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. (AU)

• Processo FAPESP: 08/57336-2 - Obtenção e caracterização das metacaspases: participação destas enzimas na morte celular
• Beneficiário: Mauricio Ferreira Marcondes Machado
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado