The Catalytic Role of Glutamate 151 in the Leucine Aminopeptidase from Aeromonas proteolytica

Krzysztof P Bzymek,Richard C Holz

doi:10.1074/jbc.m404035200

Abstract

Glutamate 151 has been proposed to act as the general acid/base during the peptide hydrolysis reaction catalyzed by the co-catalytic metallohydrolase from Aeromonas proteolytica (AAP). However, to date, no direct evidence has been reported for the role of Glu-151 during catalytic turnover by AAP. In order to elucidate the catalytic role of Glu-151, altered AAP enzymes have been prepared in which Glu-151 has been substituted with a glutamine, an alanine, and an aspartate. The Michaelis constant (K(m)) does not change upon substitution to aspartate or glutamine, but the rate of the reaction changes drastically in the following order: glutamate (100% activity), aspartate (0.05%), glutamine (0.004%), and alanine (0%). Examination of the pH dependence of the kinetic constants k(cat) and K(m) revealed a change in the pK(a) of a group that ionizes at pH 4.8 in recombinant leucine aminopeptidase (rAAP) to 4.2 for E151D-AAP. The remaining pK(a) values at 5.2, 7.5, and 9.9 do not change. Proton inventory studies indicate that one proton is transferred in the rate-limiting step of the reaction at pH 10.50 for both rAAP and E151D-AAP, but at pH 6.50 two protons and general solvation effects are responsible for the observed effects in the reaction catalyzed by rAAP and E151D-AAP, respectively. Based on these data, Glu-151 is intrinsically involved in the peptide hydrolysis reaction catalyzed by AAP and can be assigned the role of a general acid and base.

Highlights

(DapE), the argE-encoded N-acetyl-L-ornithine deacetylase (ArgE), carboxypeptidase G2, and glutamate carboxypeptidase II, sometimes referred to as N-acetylated ␣-linked acidic dipeptidase, all play important roles in living organisms (1, 2, 6 – 8)
Glutamate 151 has been proposed to act as the general acid/base during the peptide hydrolysis reaction catalyzed by the co-catalytic metallohydrolase from Aeromonas proteolytica (AAP)
Metalloproteases containing co-catalytic active sites are widely regarded as promising targets for drug discovery, but efficient drug discovery has been hampered by the lack of mechanistic detail for this class of enzymes

Summary

Introduction

(DapE), the argE-encoded N-acetyl-L-ornithine deacetylase (ArgE), carboxypeptidase G2, and glutamate carboxypeptidase II, sometimes referred to as N-acetylated ␣-linked acidic dipeptidase, all play important roles in living organisms (1, 2, 6 – 8). AAP has been crystallographically characterized and possesses a (␮-aqua)(␮carboxylato)dizinc(II) core with a terminal carboxylate and histidine residue coordinated to each metal ion [19]. Whereas AAP is not a specific target for pharmaceuticals at this time, a detailed understanding of its mechanism is still quite important with regard to other metallohydrolases that contain co-catalytic active sites that are drug targets [1,2,3]. For these reasons, AAP is one of the best mechanistically characterized hydrolytic enzymes with a co-catalytic metallo-active site [2]. Reveals that an oxygen atom of Glu-151 forms a hydrogen bond with a water molecule that bridges the two Zn(II) ions [19]

Methods

Results

Conclusion