Abstract
The aim of this work is to discover the inhibitory mechanism of tea peptides and to analyse the affinities between the peptides and the angiotensin-converting enzyme (ACE) as well as the stability of the complexes using in vitro and in silico methods. Four peptide sequences identified from tea, namely peptides I, II, III, and IV, were used to examine ACE inhibition and kinetics. The half maximal inhibitory concentration (IC50) values of the four peptides were (210.03±18.29), (178.91±5.18), (196.31±2.87), and (121.11±3.38) µmol/L, respectively. The results of Lineweaver-Burk plots showed that peptides I, II, and IV inhibited ACE activity in an uncompetitive manner, which requires the presence of substrate. Peptide III inhibited ACE in a non-competitive manner, for which the presence of substrate is not necessary. The docking simulations showed that the four peptides did not bind to the active sites of ACE, indicating that the four peptides are allosteric inhibitors. The binding free energies calculated from molecular dynamic (MD) simulation were −72.47, −42.20, −52.10, and −67.14 kcal/mol (1 kcal=4.186 kJ), respectively. The lower IC50 value of peptide IV may be attributed to its stability when docking with ACE and changes in the flexibility and unfolding of ACE. These four bioactive peptides with ACE inhibitory ability can be incorporated into novel functional ingredients of black tea.
摘要
目的
探究红茶中分离的茶叶多肽与血管紧张素转化酶(ACE)的结合关系以及结合稳定性, 从而发现红茶多肽对ACE的抑制机理.
创新点
ACE是哺乳动物调节血压的关键酶, 越来越多的研究着力于从食物蛋白中发现天然无副作用的ACE抑制剂. 计算机模拟技术具有高效、低成本、高通量等优点, 本研究从体外实验和计算机模拟两个维度来分析多肽与ACE之间的相互作用, 并使用了分子对接和分子动力学模拟两种计算机模拟方法, 使结果更加可靠.
方法
用体外酶抑制实验获取茶叶多肽(红茶中分离)对ACE的半抑制浓度(IC50);用酶动力学实验探究红茶多肽对ACE的抑制类型;通过分子对接技术预测茶叶多肽在ACE蛋白中的结合位置;通过100 ns的分子动力学模拟实验评价各多肽对ACE蛋白自由度和展开度的影响以及多肽-蛋白结合物的稳定性.
结论
四条茶叶多肽都表现出较高的体外ACE抑制活性(IC50值分别为(210.03±18.29)、(178.91±5.18)、(196.31±2.87)和(121.11±3.38) µmol/L), 酶动力学实验表明多肽I、II和IV对ACE的抑制类型为反竞争性抑制(即需要底物先与酶结合才能发挥作用), 多肽III为非竞争性抑制(即不管底物是否与酶结合都能发挥作用). 分子对接结果显示四条多肽都对接在远离ACE活性口袋的空腔中, 分子动力学模拟结果揭示多肽的ACE抑制能力与其对ACE蛋白残基自由度和蛋白展开度的影响存在相关性, 多肽IV体外抑制能力最强同时对ACE蛋白的影响最大. 本研究说明四条茶叶多肽是通过改变ACE蛋白结构来达到抑制作用, 符合变构抑制剂的作用原理.
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Acknowledgments
The research was supported by the National Key Research and Development Program of China (No. 2016YFD0200900) and the Science Technology Department of Zhejiang Province (No. 2016C02053-8), China.
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Yating LU performed the experimental research and data analysis, and wrote and edited the manuscript. Yu WANG, Danyi HUANG, and Dongmei FAN performed the data analysis. Zhuang BIAN performed the experimental research. Peng LU reviewed and edited the manuscript. Xiaochang WANG supervised the research, and reviewed and edited the manuscript. All authors have read and approved the final manuscript and, therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data.
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Yating LU, Yu WANG, Danyi HUANG, Zhuang BIAN, Peng LU, Dongmei FAN, and Xiaochang WANG declare that they have no conflict of interest.
This article does not contain any studies with human or animal subjects performed by any of the authors.
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Lu, Y., Wang, Y., Huang, D. et al. Inhibitory mechanism of angiotensin-converting enzyme inhibitory peptides from black tea. J. Zhejiang Univ. Sci. B 22, 575–589 (2021). https://doi.org/10.1631/jzus.B2000520
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DOI: https://doi.org/10.1631/jzus.B2000520
Key words
- Black tea
- Angiotensin-1-converting enzyme (ACE) inhibitory peptide
- Kinetic study
- Molecular docking
- Molecular dynamic (MD) simulation