The Structure of the <i>Aeromonas proteolytica</i> Aminopeptidase Complexed with a Hydroxamate Inhibitor

Chevrier, Bernard; d'Orchymont, Hugues; Schalk, C.; Tarnus, Céline; Moras, Dino

doi:10.1111/j.1432-1033.1996.0393k.x

Cited by 138 publications

(142 citation statements)

References 30 publications

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“…These data suggest that an enzyme residue is required to be in the protonated form for the substrate to bind. The apparent pK a value obtained suggests a tyrosine residue (pK a of 10.5), one of which (Tyr-225) was previously implicated in hydrogen bond formation with the substrate (38,42,43). The pK a value observed at pH 7.5 suggests that a protonated form of the substrate or a group on the enzyme results in decreased substrate binding affinity assisting in ES complex formation.…”

Section: Temperature Dependence Of K Cat and K M For E151d-aap-mentioning

confidence: 89%

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

Bzymek

Holz

2004

Journal of Biological Chemistry

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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.

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Section: Temperature Dependence Of K Cat and K M For E151d-aap-mentioning

confidence: 89%

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

Bzymek

Holz

2004

Journal of Biological Chemistry

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“…The PSMA models are based on sequence homologies between its catalytic domain and that of zinc aminopeptidases from Vibrio proteolyticus and Streptomyces griseus. These enzymes belong to the M28 protease family that includes PSMA, and their x-ray crystal structures have been determined (29). Both the Vibrio and Streptomyces enzymes crystallize and are enzymatically active as monomers.…”

Section: Discussionmentioning

confidence: 99%

The homodimer of prostate-specific membrane antigen is a functional target for cancer therapy

Schülke

Varlamova

Donovan

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

199

163

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Prostate-specific membrane antigen (PSMA) is a type 2 integral membrane glycoprotein that serves as an attractive target for cancer immunotherapy by virtue of its abundant and restricted expression on the surface of prostate carcinomas and the neovasculature of most other solid tumors. However, relatively little is known about the molecular structure of this target. Here, we report that PSMA is expressed on tumor cells as a noncovalent homodimer. A truncated PSMA protein, lacking transmembrane and cytoplasmic domains, also formed homodimers, indicating that the extracellular domain is sufficient for dimerization. PSMA dimers but not monomers displayed a native conformation and possessed high-level carboxypeptidase activity. A unique dimerspecific epitope was identified by using one of a panel of novel mAbs. When used to immunize animals, dimer but not monomer elicited antibodies that efficiently recognized PSMA-expressing tumor cells. These findings on PSMA structure and biology may have important implications for active and passive immunotherapy of prostate and other cancers.

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“…In the Vibrio aminopeptidase, the corresponding glutamate (Glu-151) has been proposed to be a general base in the catalytic mechanism and has been shown to be involved in binding to the bridging H 2 O or phosphate or to a hydroxamate inhibitor (Table 1) (Chevrier et al, 1996). In the mononuclear carboxypeptidase A and thermolysin, a glutamate residue, implicated in the catalytic mechanism, is in a HEXXH motif in which the Glu is the putative catalytic residue and the two His are Zn ϩϩ ligands.…”

Section: Discussionmentioning

confidence: 99%

Site-Directed Mutagenesis of Predicted Active Site Residues in Glutamate Carboxypeptidase II

et al. 1999

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Glutamate carboxypeptidase II (GCP II) catalyzes the extracellular hydrolysis of the neuromodulator N-acetyl-aspartylglutamate to N-acetyl-aspartate and glutamate. GCP II also hydrolyzes ␥-glutamyl bonds in folylpolyglutamate. The predicted amino acid sequence of GCP II displays similarities to aminopeptidases from Streptomyces griseus and Vibrio proteolyticus, whose crystal structures have been determined. These aminopeptidases are cocatalytic zinc metallopeptidases belonging to the peptidase family M28. Specific zinc and substrate ligands have been proposed in GCP II based on the amino acid sequence alignment to these M28 family members. In the present study, site-directed mutagenesis has been used to test the assignment of these putative ligands in human GCP II. Substitutions to the five putative zinc ligands resulted in severely reduced enzyme activity, although mutant protein was expressed as demonstrated by immunoblot analysis. In addition, substitutions of amino acids near the putative zinc ligands have identified other specific residues important for enzyme structure and/or function. Substitutions to putative substrate ligands were less perturbing, and increases in K m were observed for substitutions that introduced a large charge perturbation (e.g., Lys to Glu). The results from substitutions at the proposed zinc and substrate ligands are consistent with the assignment of these residues and suggest that GCP II has a three-dimensional structure similar to other members of the peptidase family M28.

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The Structure of the Aeromonas proteolytica Aminopeptidase Complexed with a Hydroxamate Inhibitor

Cited by 138 publications

References 30 publications

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

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

The homodimer of prostate-specific membrane antigen is a functional target for cancer therapy

Site-Directed Mutagenesis of Predicted Active Site Residues in Glutamate Carboxypeptidase II

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