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Identification of threonine 348 as a residue involved in aminopeptidase A substrate specificity.

Cédric Claperon 1 Inmaculada Banegas-Font 1 Xavier Iturrioz 1 Raphael Rozenfeld 1 Bernard Maigret 2 Catherine Llorens-Cortes 1 
1 Neuropeptides centraux et régulations hydrique et cardiovasculaire
UPMC - Université Pierre et Marie Curie - Paris 6, CIRB - Centre interdisciplinaire de recherche en biologie, INSERM - Institut National de la Santé et de la Recherche Médicale : U691
2 ORPAILLEUR - Knowledge representation, reasonning
INRIA Lorraine, LORIA - Laboratoire Lorrain de Recherche en Informatique et ses Applications
Abstract : Aminopeptidase A (APA; EC is a membrane-bound zinc metalloprotease cleaving in the brain the N-terminal aspartyl residue of angiotensin II to generate angiotensin III, which exerts a tonic stimulatory effect on the central control of blood pressure in hypertensive animals. We docked the specific APA inhibitor, glutamate phosphonate, in the three-dimensional model of the mouse APA ectodomain in the presence of Ca(2+). In the S1 subsite of this model, the Ca(2+) atom was coordinated with Asp-213, Asp-218,y and Glu-215 and three water molecules, one of which formed a hydrogen bond with the carboxylate side chain of the inhibitor. We report here that the carboxylate side chain of glutamate phosphonate also formed a hydrogen bond with the alcohol side chain of Thr-348. Mutagenic replacement of Thr-348 with an aspartate, tyrosine, or serine residue led to a modification of the hydrolysis velocity, with no change in the affinity of the recombinant enzymes for the substrate GluNA, either in the absence or presence of Ca(2+). In the absence of Ca(2+), the mutations modified the substrate specificity of APA, which was nevertheless restored by the addition of Ca(2+). An analysis of three-dimensional models of the corresponding Thr-348 mutants revealed that the interaction between this residue and the inhibitor was abolished or disturbed, leading to a change in the position of the inhibitor in the active site. These findings demonstrate a key role of Thr-348 in substrate specificity of APA for N-terminal acidic amino acids by insuring the optimal positioning of the substrate during catalysis.
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Submitted on : Monday, September 21, 2009 - 4:20:45 PM
Last modification on : Thursday, March 17, 2022 - 10:08:35 AM

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Cédric Claperon, Inmaculada Banegas-Font, Xavier Iturrioz, Raphael Rozenfeld, Bernard Maigret, et al.. Identification of threonine 348 as a residue involved in aminopeptidase A substrate specificity.. Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2009, 284 (16), pp.10618-26. ⟨10.1074/jbc.M806783200⟩. ⟨inserm-00418798⟩



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