首页 > 过刊浏览>2022年第41卷第5期 >169-178. DOI:10.13300/j.cnki.hnlkxb.2022.05.021
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石金钱龟板来源的寡肽与环氧合酶-2相互作用机制
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作者:
作者单位:

华中农业大学食品科学技术学院/环境食品学教育部重点实验室，武汉 430070

作者简介:

闫佳兴，E-mail：1813425989@qq.com

通讯作者:

张久亮，E-mail：zjl_ljz@mail.hzau.edu.cn

中图分类号:

TS201.2

基金项目:

国家重点研发计划项目（2021YFE0194000）


Interaction mechanism between oligopeptides from yellow pond turtle and cyclooxygenase-2 based on fluorescence spectroscopy and molecular docking
Author:
Affiliation:

College of Food Science and Technology， Huazhong Agricultural University/ Key Laboratory of Environment Correlative Dietology，Ministry of Education， Wuhan 430070， China

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    摘要:

    为探究石金钱龟板来源的寡肽KNGP能否作为COX-2（环氧合酶-2）的新型抑制剂，通过紫外分光光度法测定石金钱龟板肽YPTP、合成肽段KNGP和RG的IC50值，利用内源荧光、同步荧光、三维荧光光谱和ANS（1-苯胺基-8-萘磺酸）荧光探针法分析KNGP对COX-2的疏水性影响，利用分子对接方法分析KNGP与COX-2间的相互作用方式。结果显示，YPTP、KNGP和RG的IC50值分别为0.609、0.046和0.056 mg/mL，KNGP的IC50值低于阳性药布洛芬（IC50=0.217 mg/mL），说明KNGP对COX-2具有显著的抑制潜力。内源荧光结果显示KNGP对COX-2的作用为静态猝灭且有单一作用位点，同时热力学参数计算结果显示，KNGP和COX-2的结合过程中疏水作用是主要驱动力，且KNGP和COX-2结合的是由熵驱动的自发过程。同步荧光、三维荧光光谱和荧光探针结果显示COX-2中的酪氨酸与色氨酸的疏水环境发生变化，且其表面的疏水性减弱。KNGP与COX-2的分子对接显示，KNGP分子主要通过NH基团、C=O结构、吲哚和咪唑结构与COX-2活性中心的氨基酸残基形成氢键和疏水相互作用，并通过形成静电作用增强结合的稳定性。以上结果表明， KNGP能与COX-2的单一位点通过氢键和疏水相互作用结合形成复合物，并改变酶的二级结构来抑制其活性。

    Abstract:

    This study aimed to reveal the mechanism of action of oligopeptides KNGP on cyclooxygenase-2 （COX-2）， and to explore the interaction between KNGP from yellow pond turtle peptides （YPTP） and COX-2.The IC50 values of YPTP， synthesized KNGP and RG were determined by UV spectrophotometry.The mechanism of interaction between KNGP and COX-2 was investigated by intrinsic fluorescence， synchronous fluorescence， three-dimensional fluorescence spectroscopic techniques and ANS fluorescence probe.The mode of action between KNGP and COX-2 was studied by molecular docking.The results showed that the IC50 values of YPTP， KNGP and RG were 0.609， 0.046 and 0.056 mg/mL， respectively.The IC50 of KNGP was lower than that of the positive drug ibuprofen （IC50=0.217 mg/mL）， indicating that KNGP had a significant inhibitory effect on COX-2 enzyme.The intrinsic fluorescence results showed that KNGP had a static quenching effect on COX-2， and the action site was single.Meanwhile， the results of thermodynamic parameter calculations showed that hydrophobic interaction is the main driving force in the process of KNGP binding to COX-2， and KNGP binding to COX-2 is a spontaneous process driven by entropy.The results of synchronous fluorescence， three-dimensional fluorescence spectroscopic and ANS fluorescence probe showed that the hydrophobic environment of tyrosine and tryptophan in COX-2 changed， and the hydrophobicity of COX-2 surface was weakened.The molecular docking between KNGP and COX-2 showed that KNGP molecules formed hydrogen bonds and hydrophobic interactions with the amino acid residues of COX-2 active center mainly through NH， C=O structure， indole and imidazole structure， and enhanced the binding stability by forming electrostatic interaction.Therefore， the results showed that KNGP could form a complex with COX-2 at a single site through hydrogen bonding and hydrophobic interaction， and change the secondary structure of the enzyme to inhibit its activity.

    表 4 KNGP、阳性对照（布洛芬和对乙酰氨基酚）与COX-2的对接打分结果Table 4 Results of molecular docking between KNGP， positive controls （ibuprofen and acetaminophen） and COX-2
    表 1 不同温度下KNGP对COX-2的猝灭常数Table 1 Quenching constants of KNGP on COX-2 at three temperatures
    表 2 不同温度KNGP对COX-2的结合常数和结合位点数Table 2 Binding constant and binding number of KNGP on COX-2 at three temperatures
    图1 石金钱龟龟板肽YPTP（A）、合成肽段KNGP（B）、RG（C）和布洛芬（D）对COX-2的抑制率Fig.1 Inhibitory rates of YPTP （A）， KNGP （B）， RG （C） and ibuprofen （D） on COX-2
    图2 COX-2与不同浓度的KNGP在298 K下的内源荧光光谱Fig.2 Intrinsic fluorescence spectra of COX-2 with KNGP at 298 K
    图3 COX-2与KNGP在298、304和310 K下的Stern-Volmer猝灭曲线Fig.3 Stern-Volmer plots for the quenching of COX-2 with KNGP at 298 K， 304 K and 310 K
    图4 KNGP与COX-2的双对数回归曲线Fig.4 The plots for calculating the binding constants and the number of binding sites of KNGP to COX-2
    图5 KNGP对COX-2的同步荧光光谱Fig.5 Synchronous fluorescence spectra of COX-2 with KNGP
    图6 KNGP对COX-2的RSFQ曲线Fig.6 The corresponding plot of RSFQ of COX-2 with KNGP
    图7 COX-2（A）三维荧光等高线图和在KNGP（0.47 mmol/L） 时COX-2（B）三维荧光等高线图Fig.7 Three-dimensional fluorescence spectra of COX-2 in absence （A） and presence of KNGP （0.47 mmol/L， B）
    图8 KNGP对COX-2的ANS荧光光谱Fig.8 ANS-fluorescence spectra of COX-2 with KNGP
    图9 KNGP与COX-2分子对接ROC曲线（A）及ROC的曲线下面积（B）Fig.9 ROC curves （A） and AUC of ROC curve （B） of docking between KNGP and COX-2
    图10 KNGP与COX-2对接的活性位点和特定氨基酸残基的3D（A）和2D（B）对接结果图Fig.10 3D （A） and 2D （B） docking mode of KNGP to the active site and specific amino acid residues of COX-2
    表 3 不同温度KNGP对COX-2的热力学参数Table 3 Thermodynamic parameters of KNGP on COX-2 at three temperatures
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闫佳兴,张久亮.石金钱龟板来源的寡肽与环氧合酶-2相互作用机制[J].华中农业大学学报,2022,41(5):169-178

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