Aminopeptidase A (APA) is a membrane-bound monozinc aminopeptidase. In the brain, APA generates angiotensin III which exerts a tonic stimulatory effect on the control of blood pressure (BP) in hypertensive animals. The oral administration of RB150 renamed firibastat by WHO, an APA inhibitor prodrug, targeting only the S1 subsite, decreases BP in hypertensive patients from various ethnic origins. To identify new families of potent and selective APA inhibitors, we explored the organization of the APA active site, especially the S2′ subsite. By molecular modeling, docking, molecular dynamics simulations and site-directed mutagenesis, we revealed that Arg368 and Arg386, in the S2′ subsite of human APA established various types of interactions in major part with the P2′ residue but also with the P1′ residue of APA inhibitors, required for their nanomolar inhibitory potency. We also demonstrated an important role for Arg368 in APA catalysis, in maintaining the structural integrity of the GAMEN motif, a conserved sequence involved in exopeptidase specificity and optimal positioning of the substrate in monozinc aminopeptidases. This arginine together with the GAMEN motif are key players for the catalytic mechanism of these enzymes.

中文翻译：

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对氨基肽酶A催化机理和抑制剂结合的结构见解

氨肽酶A（APA）是膜结合的单锌氨基肽酶。在大脑中，APA产生血管紧张素III，对高血压动物的血压（BP）的控制产生补益刺激作用。WHO口服的RB150重命名为非ibstatin，这是一种APA抑制剂前药，仅针对S1亚位点，可降低来自不同种族的高血压患者的BP。为了确定有效和选择性APA抑制剂的新家族，我们探索了APA活性位点，特别是S2'亚位点的组织。通过分子建模，对接，分子动力学模拟和定点诱变，我们发现人APA的S2'亚位的Arg368和Arg386主要与P2'残基也与P1'残基建立了各种类型的相互作用APA抑制剂 所需的纳摩尔抑制力。我们还证明了Arg368在APA催化中的重要作用，可保持GAMEN基序的结构完整性，参与肽外切酶特异性的保守序列和单锌氨肽酶中底物的最佳定位。精氨酸与GAMEN基序一起是这些酶催化机制的关键角色。