Skip to main content
Article thumbnail
Location of Repository

Identification of Threonine 348 as a Residue Involved in Aminopeptidase A Substrate Specificity*

By Cédric Claperon, Inmaculada Banegas-Font, Xavier Iturrioz, Raphael Rozenfeld, Bernard Maigret and Catherine Llorens-Cortes


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 Ca2+. In the S1 subsite of this model, the Ca2+ 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 Ca2+. In the absence of Ca2+, the mutations modified the substrate specificity of APA, which was nevertheless restored by the addition of Ca2+. 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

Topics: Enzyme Catalysis and Regulation
Publisher: American Society for Biochemistry and Molecular Biology
OAI identifier:
Provided by: PubMed Central
Download PDF:
Sorry, we are unable to provide the full text but you may find it at the following location(s):
  • http://www.pubmedcentral.nih.g... (external link)
  • Suggested articles

    To submit an update or takedown request for this paper, please submit an Update/Correction/Removal Request.