inserm-00418798, version 1

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

The Journal of Biological Chemistry 284, 16 (2009) 10618-26

• 1 :  Neuropeptides centraux et régulations hydrique et cardiovasculaire
• 
  INSERM : U691 – Collège de France – Université Pierre et Marie Curie [UPMC] - Paris VI 11, Place Marcelin Berthelot 75231 Paris Cedex 05 France
• 2 :  ORPAILLEUR (INRIA Lorraine - LORIA)
• 
  INRIA – CNRS : UMR7503 – Université Henri Poincaré - Nancy I – Université Nancy II – Institut National Polytechnique de Lorraine (INPL) France

Références bibliographiques

• Type de publication : Articles dans des revues avec comité de lecture
• PMID: identifiant
  de la référence Pubmed : (19228697)
• titre : Identification of threonine 348 as a residue involved in aminopeptidase A substrate specificity.
• résumé : Aminopeptidase A (APA; EC 3.4.11.7) 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.
• domaine : Sciences du Vivant/Médecine humaine et pathologie/Endocrinologie et métabolisme
• langue du texte
  intégral : Anglais
• ISSN : 0021-9258
• DOI : 10.1074/jbc.M806783200
• journal : The Journal of Biological Chemistry
• Audience : internationale
• date de publication : 17/04/2009
• date de publication
  électronique : 19/02/2009
• volume : 284
• numéro : 16
• page, identifiant, ... : 10618-26
• Descripteur(s) MeSH : Adenosine – Animals – Arvicolinae – Blotting – Western – CHO Cells – Calcium – Cell Line – Cricetinae – Humans – Kidney – Microscopy – Confocal – Protein Binding – RNA Interference – RNA – Small Interfering – Radioligand Assay – Receptor – Adenosine A2B – Adenosine A3 – Receptors – G-Protein-Coupled – Ghrelin – Transfection – Angiotensin III – Glutamates – Glutamyl Aminopeptidase – Mice – Molecular Conformation – Molecular Structure – Mutagenesis – Site-Directed – Phosphonic Acids – Recombinant Proteins – Substrate Specificity – Threonine

• inserm-00418798, version 1
• http://www.hal.inserm.fr/inserm-00418798
• oai:www.hal.inserm.fr:inserm-00418798
• Contributeur : Catherine Llorens Cortes <>
• Soumis le : Lundi 21 Septembre 2009, 16:20:45
• Dernière modification le : Jeudi 8 Octobre 2009, 10:18:48