Contribution of Molecular Modeling and Site-Directed Mutagenesis to the Identification of a New Residue, Glutamate 215, Involved in the Exopeptidase Specificity of Aminopeptidase A - CNRS - Centre national de la recherche scientifique Accéder directement au contenu
Article Dans Une Revue Biochemistry Année : 2003

Contribution of Molecular Modeling and Site-Directed Mutagenesis to the Identification of a New Residue, Glutamate 215, Involved in the Exopeptidase Specificity of Aminopeptidase A

Raphaël Rozenfeld
  • Fonction : Auteur
Mayumi Okada
  • Fonction : Auteur
Bernard Maigret
  • Fonction : Auteur
Catherine Llorens-Cortes

Résumé

Aminopeptidase A is a zinc metalloenzyme that generates brain angiotensin III, which exerts a tonic stimulatory action on blood pressure in hypertensive animals. We have previously constructed a three-dimensional model of the ectodomain of this enzyme, using the crystal structure of leukotriene A4 hydrolase/aminopeptidase as a template. According to this model, Glu-215, which is located in the active site, hydrogen bonds to the amino moiety of the inhibitor, 4-amino-4-phosphonobutyric acid (GluPhos), a phosphonic acid anologue of glutamic acid. Replacement of this residue with an aspartate or an alanine in the model abolished this interaction and led to a change in the position of the inhibitor in the active site. Mutagenic replacement of Glu-215 with an aspartate or an alanine drastically reduced the affinity of the recombinant enzymes for the substrate by a factor of 10 or 17, respectively, and the rate of hydrolysis by a factor of 14 or 6, respectively. Two isomers of GluPhos with different N-terminal amine positions differed considerably in their ability to inhibit the wild type (by a factor of 40), but not the mutated enzymes. These results, together with the interaction predicted by the model, demonstrate that Glu-215 interacts with the N-terminal amine of the substrate, thereby contributing, together with Glu-352, to the determination of the exopeptidase specificity of aminopeptidase A.

Dates et versions

hal-03631059 , version 1 (05-04-2022)

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Citer

Raphaël Rozenfeld, Xavier Iturrioz, Mayumi Okada, Bernard Maigret, Catherine Llorens-Cortes. Contribution of Molecular Modeling and Site-Directed Mutagenesis to the Identification of a New Residue, Glutamate 215, Involved in the Exopeptidase Specificity of Aminopeptidase A. Biochemistry, 2003, 42 (50), pp.14785-14793. ⟨10.1021/bi034358u⟩. ⟨hal-03631059⟩
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