Aeromonas proteolytica Aminopeptidase:  An Investigation of the Mode of Action Using a Quantum Mechanical/Molecular Mechanical Approach

Abstract

The aminopeptidase of Aeromonas proteolytica (AAP) belongs to the group of metallo-hydrolases that require two divalent cations for full activity. Such binuclear metal centers are found in several aminopeptidases, raising the question whether a common mechanism, at least partly, is likely. We have used a quantum mechanical/molecular mechanical (QM/MM) approach to investigate the reaction mechanism of AAP. Among several possibilities, one reaction path was found to be clearly the most favorable. Beside the chemical transformation steps, effects of the enzyme environment and the influence of the solvent on the catalytic reaction were included in the study. The results are in good agreement with experimental studies and correspond to a high degree to our previous QM/MM calculations on the reaction mechanism of the related binuclear bovine lens leucine aminopeptidase (blLAP), which, although related to the AAP, has different Zn(2+)-coordination spheres and a different catalytic residue. The mechanisms of the two enzymes as suggested in the literature differ on the mode of coordination of the nucleophile and the identity of the general base. However, the results of this and our previous work on blLAP allow us to identify a common mechanism for the two enzymes. This mechanism is probably quite general for binuclear zinc enzymes.

Crystal Structure of TET Protease Reveals Complementary Protein Degradation Pathways in Prokaryotes

“…Recent studies propose a common mechanism of peptide cleavage for the binuclear metallo-aminopeptidases AAP and LAP. 24 We confirm that TET follows this mechanism, in which the first step of catalysis is performed by the active site nucleophilic water molecule, attacking the scissile carbonyl carbon atom of the substrate between P1 and P1 0 . The binding mode of amastatin represents the tetrahedral intermediate in the enzyme-catalysed peptide hydrolysis.…”

“…17,18 The crystal structure of TET in complex with amastatin allows us to propose that TET follows the common mechanism of hydrolysis displayed by these aminopeptidases. 24 The insight for the broad substrate specificity of TET comes from the arrangement of residues forming the specificity pockets. The coordination of the substrate peptide is achieved only at the P1 and P1 0 sites.…”

X-ray crystallographic characterization of the Co(II)-substituted Tris-bound form of the aminopeptidase from Aeromonas proteolytica

“…However, this being the more strongly metal-coordinating oxygen of Glu152, a hydrogen bond with either of the expectedly more basic Glu151 oxygens appears much more likely. At an O-O distance of 2.7 Å , this would indicate a very strong hydrogen bond, where either of the involved oxygens might be protonated, and would thus be supportive of the general acid/base role that Glu151 has been proposed to play at the various stages of the reaction mechanism [31,38]. Based on the coordination distances of O4 from the metals, one could postulate O4 to be protonated in the Co-loaded complex and deprotonated in the Zn-loaded one.…”

The roles of glutathione peroxidases during embryo development

“…Moreover, in contrast to the other GPx family members GPx4 exhibits a broad affinity toward its substrate hydroperoxides as well as its reducing equivalents. GPx4 shares the structural preconditions for its enzymatic activity with the other GPx isoforms (Scheerer et al, 2007). In contrast to other GPx isoforms however, the active site of GPx4 is not framed by an exposed surface loop that potentially limits the accessibility of the active site and its largely hydrophobic surface allow GPx4 to closely associate with membranes and lipoproteins.…”