TEM-1 β-内酰胺酶与三种β-内酰胺类抗生素的分子识别和相互作用研究

发布时间：2018-06-07 16:39

本文选题：TEM-1 + β-内酰胺酶　；参考：《西北大学》2016年博士论文

【摘要】：抗生素耐药问题是目前人类医疗活动中面临的重大社会问题,解决这一问题的主要途径包括抗生素的合理使用、新型抗生素的研发和抗生素与其体内靶点的相互作用研究等。p-内酰胺类抗生素是临床上最常用的抗菌药物,其产生耐药性的主要原因在于：细菌编码产生抗药靶蛋白p-内酰胺酶,催化水解p-内酰胺类抗生素。TEM-1β-内酰胺酶(TEM-1)是最常见的超广谱p-内酰胺酶,其催化水解β-内酰胺类抗生素的首要环节是识别并结合抗生素。本论文选择青霉素G、头孢氨苄和头孢西丁三种β-内酰胺类抗生素为对象,首先采用位点靶向性分子模拟技术研究TEM-1与这三种抗生素的分子识别,并应用荧光光谱法和亲和色谱法研究它们相互作用的机理。该研究能从分子水平上阐明TEM-1与β-内酰胺类抗生素的相互作用机制,对解决抗生素耐药问题和研制新型高效抗生素药物具有重要意义。全文共分3章,主要工作和内容如下：1.采用分子对接技术对TEM-1与青霉素G、头孢氨苄和头孢西丁的分子识别过程进行了模拟,并采用光谱法探查TEM-1与三种抗生素发生分子识别时该酶自身构象的变化,证明分子识别是其相互作用的关键环节。结果表明：TEM-1分子和三种抗生素分子分别形成6,5和3个氢键,通过氢键生成1：1的复合物；TEM-1与三种抗生素相互作用的主要推动力是氢键和范德华力。紫外吸收光谱结果表明：随着抗生素浓度的增大,紫外吸收值增大,吸收峰红移。碘化钾(KI)猝灭实验结果表明：KI对TEM-1-抗生素复合物的猝灭常数高于对TEM-1的猝灭常数,且形成复合物后,TEM-1表面的发色氨基酸残基的数目减少。同步荧光光谱结果表明：在Λλ=60 nm时和Λλ=15 nm时,随着抗生素浓度的逐渐增大,TEM-1的同步荧光强度逐渐下降,其Λλ=60 nm时下降更显著。二阶导数荧光光谱结果表明：激发波长为278 nm时,酶与酶-抗生素复合物均有两个负峰,形成复合物后峰强度和位移均有变化：激发波长为295 nm时,酶与酶-抗生素复合物均只有一个负峰,形成复合物后峰强度降低。2.采用荧光光谱法和亲和色谱法研究TEM-1与三种抗生素分子相互作用的机制。结果表明,TEM-1与三种抗生素以1：1的比例发生相互作用,猝灭机制为静态猝灭。热力学研究结果表明：焓变ΛH、熵变AS和自由能变化ΛG均0,说明TEM-1与三种抗生素的作用过程为自发的放热过程,主要作用力为氢键和范德华力。另一方面,TEM-1和三种抗生素相互作用的表观活化能均为负值,表明结合过程没有能垒,是一个自发的过程。根据Forster能量转移理论计算得到TEM-1与三种抗生素的结合距离r均小于7 nm,且满足0.5R01.5R0,说明能量转移过程为非辐射转移。亲和色谱法的研究结果表明,固定化TEM-1仍然保留其生物活性,能特异性的识别三种抗生素分子,两者相互作用过程符合单朗格缪尔模型。前沿色谱分析测定TEM-1与青霉素G、头孢氨苄和头孢西丁相互作用过程的结合常数分别为：3.54×104 M-1,1.89×104 M-1和1.23×104 M-1,结合常数大小顺序为青霉素G头孢氨苄头孢西丁,与荧光光谱法测定结果一致,证明固定化TEM-1可用于研究TEM-1与抗生素类药物的相互作用。
[Abstract]:The problem of antibiotic resistance is a major social problem in human medical activities. The main ways to solve this problem include the rational use of antibiotics, the development of new antibiotics and the study of the interaction between the antibiotics and the targets in the body..p- lactam antibiotics are the most commonly used antibiotics in the bed, and they produce drug resistance. The main reason is that bacteria encode the production of anti drug target protein p- lactamase, and catalyze the hydrolysis of p- lactam antibiotic.TEM-1 beta lactamase (TEM-1) as the most common super broad-spectrum p- lactamase. The first link to catalyze the hydrolysis of beta lactam antibiotics is to identify and bind antibiotics. This paper chooses penicillin G, cefampicin Three kinds of beta lactam antibiotics, cefoxitin, are used to study the molecular recognition of TEM-1 and the three antibiotics using site targeting molecular simulation technique. The mechanism of their interaction is studied by fluorescence spectroscopy and affinity chromatography. This study can elucidate the phase of TEM-1 and beta lactam antibiotics at the molecular level. The interaction mechanism is of great significance for the solution of antibiotic resistance and the development of new efficient antibiotic drugs. The full text is divided into 3 chapters. The main work and contents are as follows: 1. the molecular recognition process of TEM-1 and penicillin G, cefamaxin and cefoxitin was simulated by molecular docking, and three species were detected by spectral method. The change in the conformation of the enzyme at the time of molecular recognition shows that molecular recognition is the key link in its interaction. The results show that TEM-1 and three kinds of antibiotic molecules form 6,5 and 3 hydrogen bonds respectively, and the complex of 1:1 through hydrogen bonds; the main driving force for the interaction between TEM-1 and three kinds of antibiotics is hydrogen bond and Fan De The UV absorption spectra showed that the UV absorption value increased with the increase of antibiotic concentration, and the absorption peak was red shift. The results of the quenching experiment of potassium iodide (KI) showed that the quenching constant of KI to TEM-1- antibiotic complex was higher than that of the quenching constant to TEM-1, and the number of amino acid residues on the TEM-1 surface decreased after the formation of complex. The results of the step fluorescence spectrum showed that the synchronous fluorescence intensity of TEM-1 decreased gradually with the increasing of the concentration of the antibiotics at the lambda =60 nm and the lambda =15 nm. The two order derivative fluorescence spectra showed that when the excitation wavelength was 278 nm, the enzyme and the enzyme antibiotic complex had two negative peaks, forming a complex. The intensity and displacement of the post peak were changed: when the excitation wavelength was 295 nm, the enzyme and enzyme antibiotic complex had only one negative peak, and the peak intensity of the complex was reduced by.2.. The interaction mechanism between TEM-1 and three kinds of antibiotic molecules was studied by fluorescence spectroscopy and affinity chromatography. The results showed that the proportion of TEM-1 and three antibiotics was in the proportion of 1:1. The quenching mechanism is static quenching. The thermodynamic study shows that the enthalpy change a H, the entropy change AS and the free energy change a G are all 0, indicating that the action process of the TEM-1 and the three kinds of antibiotics is spontaneous exothermic process, the main force is hydrogen bond and Fan Dehua force. On the other hand, the apparent activation energy of the interaction of TEM-1 and three kinds of antibiotics are all Negative value indicates that the binding process has no energy barrier and is a spontaneous process. According to the Forster energy transfer theory, the binding distance between TEM-1 and three antibiotics is less than 7 nm and satisfies 0.5R01.5R0, indicating that the energy transfer process is non radiative transfer. The results of affinity chromatography still retain the biological activity of immobilized TEM-1. The interaction process of three kinds of antibiotic molecules can be specifically identified. The interaction process of the two is consistent with the single Langmuir model. The binding constants of the interaction process between TEM-1 and penicillin G, cefamaxin and cefoxitin are respectively: 3.54 x 104 M-1,1.89 x 104 M-1 and 1.23 x 104 M-1, and the binding constant size sequence is penicillin G head Ceftazidime, which is consistent with the results of fluorescence spectrometry, proves that immobilized TEM-1 can be used to study the interaction between TEM-1 and antibiotics.
【学位授予单位】：西北大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R978.1

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