Identification of threonine 348 as a residue involved in aminopeptidase A substrate specificity.
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.
Claperon
Cédric
C
INSERM, U691, College de France, Paris FR-75005, France.
Banegas-Font
Inmaculada
I
Iturrioz
Xavier
X
Rozenfeld
Raphael
R
Maigret
Bernard
B
Llorens-Cortes
Catherine
C
D000805:Angiotensin III / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D005971:Glutamates / Q000737:chemistry / Q000378:metabolism; D043384:Glutamyl Aminopeptidase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D008968:Molecular Conformation; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D063065:Organophosphonates / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity; D013912:Threonine / Q000737:chemistry / Q000378:metabolism*
D005971:Glutamates; D063065:Organophosphonates; D011994:Recombinant Proteins; C111969:glutamate phosphonic acid; D000805:Angiotensin III; D013912:Threonine; D043384:Glutamyl Aminopeptidase; D002118:Calcium