Catalytic properties of thimet oligopeptidase H600A mutant

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 His600. In the present work, the role of His600 of TOP was investigated by site-directed mutagenesis. TOP H600A mutant was characterized through analysis of S1 and S1Œ specificity, pH-activity profile and inhibition by JA-2. Results showed that TOP His600 residue makes important interactions with the substrate, supporting the prediction that His600 moves toward the substrate due to a hinge movement similar to the Dcp and ACE-2. Furthermore, the mutation H600A affected both Km and kcat, showing the importance of His600 for both substrate binding and/or product release from active site. Changes in the pH-profile may indicate also the participation of His600 in TOP catalysis, transferring a proton to the newly generated NH2-terminus or helping Tyr605 and/or Tyr612 in the intermediate oxyanion stabilization.