Identification of threonine 348 as a residue involved in aminopeptidase A substrate specificity. - Archive ouverte HAL Access content directly
Journal Articles Journal of Biological Chemistry Year : 2009

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

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Abstract

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.

Dates and versions

inserm-00418798 , version 1 (21-09-2009)

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Cite

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, 2009, 284 (16), pp.10618-26. ⟨10.1074/jbc.M806783200⟩. ⟨inserm-00418798⟩
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