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Characterization of Glu350 as a critical residue involved in the N-terminal amine binding site of aminopeptidase N (EC 3.4.11.2): insights into its mechanism of action
- Source :
- Biochemistry, Biochemistry, 1998, 37 (2), pp.686-92. ⟨10.1021/bi971705p⟩, Biochemistry, American Chemical Society, 1998, 37 (2), pp.686-92. ⟨10.1021/bi971705p⟩, Biochemistry, American Chemical Society, 1998, 37 (2), pp.686-92. 〈10.1021/bi971705p〉
- Publication Year :
- 1998
-
Abstract
- The molecular components ensuring the strict exopeptidase action of aminopeptidase N (APN) and related zinc aminopeptidases of the M1 family have not yet been clearly established. The specific recognition of the N-terminal amino acid of the substrates by the enzymes has been proposed to involve either the complexation of the free amino group by the catalytic zinc ion or an interaction with an anionic binding site, which could be constituted by an aspartate or glutamate residue. To investigate the existence of such an ionic binding site, site-directed mutagenesis experiments have been performed on acidic residues of pig APN. Given that aminopeptidases of the M1 family are likely to have a common mechanism of action, only strictly conserved residues were mutated. As compared to the wild-type enzyme, the mutation D220E led only to slight modifications in the kinetic parameters of the enzyme and in the Ki values of various inhibitors, indicating that this residue is not critically involved in the hydrolytic mechanism. In contrast, the mutations E350Q and E350D induced a large decrease in enzyme activity, essentially due to modifications in kcat, whereas the E350A mutation led to an almost completely inactive enzyme. Moreover, among the inhibitors tested, only those acting as transition state analogs showed significant increases in their Ki values. These data are in favor of E350 belonging to the "anionic binding site" in APN. A mechanism of action, derived from that of thermolysin, is proposed for these aminopeptidases, which explains the importance of E350 in transition state formation, rather than in the Michaelis complex.
- Subjects :
- Swine
MESH: Amino Acid Sequence
Biochemistry
0302 clinical medicine
MESH: Animals
chemistry.chemical_classification
0303 health sciences
MESH: Protease Inhibitors
biology
MESH: Antigens, CD13
MESH: Models, Chemical
Amine binding
MESH: Glutamic Acid
Recombinant Proteins
Amino acid
MESH: COS Cells
MESH: Mutagenesis, Site-Directed
030220 oncology & carcinogenesis
COS Cells
medicine.symptom
Protein Binding
MESH: Mutation
Stereochemistry
MESH: Peptide Hydrolases
Molecular Sequence Data
chemistry.chemical_element
Glutamic Acid
Zinc
CD13 Antigens
03 medical and health sciences
Residue (chemistry)
Exopeptidases
medicine
[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology
MESH: Protein Binding
Animals
MESH: Exopeptidases
[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology
Protease Inhibitors
Amino Acid Sequence
Binding site
030304 developmental biology
MESH: Molecular Sequence Data
Binding Sites
Sequence Homology, Amino Acid
Exopeptidase
Enzyme
Mechanism of action
chemistry
MESH: Binding Sites
Models, Chemical
MESH: Recombinant Pr
Mutation
biology.protein
Mutagenesis, Site-Directed
Peptide Hydrolases
Subjects
Details
- ISSN :
- 00062960 and 15204995
- Volume :
- 37
- Issue :
- 2
- Database :
- OpenAIRE
- Journal :
- Biochemistry
- Accession number :
- edsair.doi.dedup.....cd4139af3b80db18319e9686d038a632