Aminopeptidase A (EC 3.4.11.7, APA) is a 130 kDa membrane-bound aminopeptidase that contains the consensus sequence HEXXH (385–389) found in the zinc metalloprotease family, the zincins. Sequence alignment of the mouse APA with other monozinc-aminopeptidases indicates the presence of a highly conserved glutamate residue (Glu352 in APA) found in the conserved motif GAMEN (349–353). In monozinc-aminopeptidases, the negative charge of the glutamate side-chain carboxylate may constitute the anionic binding site involved in the recognition of the free amino group of substrates or inhibitors. The functional role of Glu352 in APA was investigated by substituting this residue with an aspartate (Asp352), a glycine (Gly352), a glutamine (Gln352) or an arginine (Arg352) residue by site-directed mutagenesis. Kinetic studies showed that the Km values of the mutant enzymes were unaffected, whereas kcat values were decreased 10–250-fold, resulting in a 10-, 30- 260- and 400-fold reduction in cleavage efficiencies for the mutants Asp352, Gly352, Gln352 and Arg352 respectively. The inhibitory potency of two different classes of inhibitors, a thiol and a phosphonate compound, was significantly (P< 0.05) decreased by 10- and 4-fold respectively in the mutated enzymes. Moreover, the inhibitory potency of angiotensin I, used as a competitor of the synthetic substrate α-l-glutamyl β-naphthylamide, displayed a 4-fold reduction (P< 0.01) in the mutated enzymes, whereas the Ki values of its N-acetyl derivative were unchanged. These data strongly suggest that Glu352 is involved in the catalytic process of APA and contributes to the exopeptidase activity of this enzyme through interaction with the N-terminal part of substrates or inhibitors.
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September 1998
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Research Article|
September 01 1998
A glutamate residue contributes to the exopeptidase specificity in aminopeptidase A
Gilles VAZEUX;
Gilles VAZEUX
1
1INSERM Unité 36, Collège de France, 3 rue d'Ulm, 75005 Paris, France
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Xavier ITURRIOZ;
Xavier ITURRIOZ
1
1INSERM Unité 36, Collège de France, 3 rue d'Ulm, 75005 Paris, France
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Pierre CORVOL;
Pierre CORVOL
1INSERM Unité 36, Collège de France, 3 rue d'Ulm, 75005 Paris, France
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Catherine LLORENS-CORTES
Catherine LLORENS-CORTES
2
1INSERM Unité 36, Collège de France, 3 rue d'Ulm, 75005 Paris, France
2To whom correspondence should be addressed (e-mail llorens@infobiogen.fr).
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Publisher: Portland Press Ltd
Received:
February 16 1998
Revision Received:
May 28 1998
Accepted:
June 30 1998
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1998
1998
Biochem J (1998) 334 (2): 407–413.
Article history
Received:
February 16 1998
Revision Received:
May 28 1998
Accepted:
June 30 1998
Citation
Gilles VAZEUX, Xavier ITURRIOZ, Pierre CORVOL, Catherine LLORENS-CORTES; A glutamate residue contributes to the exopeptidase specificity in aminopeptidase A. Biochem J 1 September 1998; 334 (2): 407–413. doi: https://doi.org/10.1042/bj3340407
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