Catalytic properties of thimet oligopeptidase H600A mutant
- Departamento de Biofisica, Universidade Federal de Sao Paulo, 04044-020 Sao Paulo, SP (Brazil)
- Laboratorio Especial de Toxinologia Aplicada, Instituto Butantan, 05467-010 Sao Paulo, SP (Brazil)
- Departamento de Biologia Celular e Desenvolvimento, Universidade de Sao Paulo, 05508-900 Sao Paulo, SP (Brazil)
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{sup 600}. In the present work, the role of His{sup 600} of TOP was investigated by site-directed mutagenesis. TOP H600A mutant was characterized through analysis of S{sub 1} and S{sub 1}' specificity, pH-activity profile and inhibition by JA-2. Results showed that TOP His{sup 600} residue makes important interactions with the substrate, supporting the prediction that His{sup 600} moves toward the substrate due to a hinge movement similar to the Dcp and ACE-2. Furthermore, the mutation H600A affected both K{sub m} and k{sub cat}, showing the importance of His{sup 600} for both substrate binding and/or product release from active site. Changes in the pH-profile may indicate also the participation of His{sup 600} in TOP catalysis, transferring a proton to the newly generated NH{sub 2}-terminus or helping Tyr{sup 605} and/or Tyr{sup 612} in the intermediate oxyanion stabilization.
- OSTI ID:
- 22202445
- Journal Information:
- Biochemical and Biophysical Research Communications, Vol. 394, Issue 2; Other Information: Copyright (c) 2010 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA); ISSN 0006-291X
- Country of Publication:
- United States
- Language:
- English
Similar Records
Crystal Structure and Conformational Dynamics of Pyrococcus furiosus Prolyl Oligopeptidase
Mammalian mitochondrial intermediate peptidase: Structure/function analysis of a new homologue from Schizophyllum commune and relationship to thimet oligopeptidases