Aminopeptidase B (APB, EC 3.4.11.6) preferentially hydrolyzes basic amino acids of synthetic substrates and requires a physiological concentration of chloride anions for optimal activity. Several amino acid residues of APB responsible for its enzymatic activity have been elucidated. In this study, we further searched for residues critical to its enzymatic activity, especially toward peptide substrates. APB residues Tyr409 (Y409) and Tyr414 (Y414), both of which were critical to its hydrolytic activity toward synthetic substrates, were predicted by molecular modeling to be involved in cleaving peptide substrates via its interaction with amino acids in the P1' cleavage site. Using site-directed mutagenesis, several mutant APBs were prepared. In contrast to synthetic substrates, wild-type and Y409F/Y414F double mutant enzymes showed P1'-dependent cleavage of peptide substrates, indicating that both tyrosine residues were not indispensable for hydrolytic activity toward peptide substrates. Moreover, the Y409F/Y414F double mutant enzyme cleaved peptides with a Pro residue at the P1' site, which is uncommon among the M1 family of aminopeptidases. These results suggested that Tyr409 and Tyr414 of APB play important roles in enzymatic function and characteristic properties of APB via proper formation of the S1' site.

中文翻译：

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Tyr409和Tyr414在构建人氨肽酶B的底物口袋中的重要性。

氨肽酶B（APB，EC 3.4.11.6）优先水解合成底物的碱性氨基酸，并且需要生理浓度的氯离子才能达到最佳活性。已经阐明了负责其酶促活性的APB的几个氨基酸残基。在这项研究中，我们进一步搜索了对其酶活性（尤其是对肽底物）至关重要的残基。通过分子模型预测，APB残基Tyr409（Y409）和Tyr414（Y414）都是其对合成底物的水解活性至关重要的残基，它们通过与P1'切割位点中的氨基酸相互作用，参与裂解肽底物。使用定点诱变，制备了几种突变型APB。与合成底物相反，野生型和Y409F / Y414F双突变酶显示P1' -依赖性的肽底物的裂解，表明酪氨酸残基对于肽底物的水解活性不是必不可少的。此外，Y409F / Y414F双突变酶切割的肽在P1'位点带有Pro残基，这在M1氨基肽酶家族中并不常见。这些结果表明，APB的Tyr409和Tyr414通过适当形成S1'位点在APB的酶功能和特性中起重要作用。