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.
Highlights
10618 JOURNAL OF BIOLOGICAL CHEMISTRY of intestinal and renal epithelial cells, and in the vascular endothelium [5]
Analysis of the Aminopeptidase A (APA) three-dimensional model complexed with glutamate phosphonate showed that Tyr-471 interacted with the inhibitor, providing support for previous demonstrations that this residue is essential for stabilizing the transition state of catalysis in APA [19]
The coverslips three-dimensional model of wild type APA complexed with were washed three times with cold PBS and incubated with glutamate phosphonate obtained in the absence of Ca2ϩ a 1:1000 dilution of cyanin 3-conjugated polyclonal anti-rabbit showed the presence of a hydrophilic pocket containing two antibody in 0.5% bovine serum albumin (BSA) in PBS for 2 h at room temperature
Summary
Restriction endonucleases and DNA-modifying enzymes were obtained from New England Biolabs Inc. and were used according to the manufacturer’s instructions. The Expand high fidelity Taq polymerase PCR system was purchased from Roche Applied Science. The liposomal transfection reagent, Lipofectamine 2000, the pcDNA 3.1-His vector, and the monoclonal anti-Xpress antibody were purchased from Invitrogen. The monoclonal anti-␣-tubulin antibody and the horseradish peroxidase-conjugated goat antimouse antibody was purchased from Sigma-Aldrich. Immobilized cobalt affinity columns (Talon) were obtained from Clontech (Heidelberg, Germany). The synthetic substrate, ␣-L-glutamyl-naphthylamide (GluNA), was purchased from Bachem (Bunderdorf, Switzerland)
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