| First Author | Claperon C | Year | 2009 |
| Journal | J Biol Chem | Volume | 284 |
| Issue | 16 | Pages | 10618-26 |
| PubMed ID | 19228697 | Mgi Jnum | J:149255 |
| Mgi Id | MGI:3848107 | Doi | 10.1074/jbc.M806783200 |
| Citation | Claperon C, et al. (2009) Identification of threonine 348 as a residue involved in aminopeptidase a substrate specificity. J Biol Chem 284(16):10618-26 |
| abstractText | 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. |
