Involvement of Phenylalanine 297 in the Construction of the Substrate Pocket of Human Aminopeptidase B.
Involvement of Phenylalanine 297 in the Construction of the Substrate Pocket of Human Aminopeptidase B.
复制标题
苯丙氨酸 297 参与人氨肽酶 B 底物袋的构建。
DOI：
10.1021/acs.biochem.5b00964
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发表时间：
2015
期刊：
影响因子：
2.9
通讯作者：
Tsujimoto M.
中科院分区：
文献类型：
--
作者：
Ohnishi A.;Watanabe J.;Ogawa Y.;Goto Y.;Hattori A.;Tsujimoto M.
氨基肽酶B （APB, EC 3.4.11.6）优先水解合成底物和肽底物的n端碱性氨基酸，需要生理浓度的NaCl才能达到最佳活性。在这项研究中，我们使用定点诱变和分子模型来寻找对人类APB酶特性至关重要的氨基酸残基。用Tyr取代Phe297导致对合成底物和肽底物的水解活性以及氯阴离子敏感性显著降低。分子模拟表明，Phe297参与了APB底物口袋的构建，该口袋的宽度足以容纳氯阴离子，并允许Gln169与底物的n端Arg残基通过桥接与氯阴离子相互作用。这些结果表明，Phe297通过形成最佳的催化袋结构，对APB的最佳酶活性和氯阴离子敏感性至关重要。
Aminopeptidase B (APB, EC 3.4.11.6) preferentially hydrolyzes the N-terminal basic amino acids of synthetic and peptide substrates and requires a physiological concentration of NaCl for optimal activity. In this study, we used site-directed mutagenesis and molecular modeling to search for an amino acid residue that is critical for the enzymatic properties of human APB. Substitution of Phe297 with Tyr caused a significant decrease in hydrolytic activity toward synthetic and peptide substrates as well as chloride anion sensitivity. Molecular modeling suggests that Phe297 contributes to the construction of the substrate pocket of APB, which is wide enough to hold a chloride anion and allow the interaction of Gln169 with the N-terminal Arg residue of the substrate through bridging with the chloride anion. These results indicate that Phe297 is crucial for the optimal enzymatic activity and chloride anion sensitivity of APB via formation of the optimal structure of the catalytic pocket.