基于别构蛋白构建智能人工抗氧化酶

发布时间：2018-05-26 11:18

本文选题：谷胱甘肽过氧化物酶 + 别构蛋白　；参考：《吉林大学》2017年博士论文

【摘要】：细胞有氧呼吸产生的活性氧自由基(Reactive Oxygen Species,ROS)会引起机体内生物大分子的损伤,进而导致白内障、克山病、阿兹海默症、心源性休克以及动脉粥样硬化等一系列重大疾病。谷胱甘肽过氧化物酶(GPx)能够以谷胱甘肽分子(GSH)为底物,利用其催化中心硒代半胱氨酸(Sec)催化过氧化物的还原,从而保护生物大分子免受ROS的损伤。鉴于GPx具有出色的抗氧化效果,人工模拟GPx成为了仿酶领域的热点之一。尤其是在催化反应活力方面,接近于天然酶的催化效率一直以来都是科学家们追求的目标。从小分子的GPx模型,到含硒抗体、半合成酶、生物印迹酶以及蛋白质GPx酶模型,大量具有高活力的人工GPx模拟酶被构建出来。这些成果对于疾病防治、延缓衰老等领域有着重要意义。除了具有极高的催化效率,严格的调控机制也是天然酶的重要特征之一。ROS并非一直都是有害的,在正常生理条件下,它们在细胞信号转导方面起着重要作用。生物体内的稳态是由多种机制共同维持的,如果单纯地服用高效的抗氧化药物,必将导致机体的代谢系统失调,反而不利于正常的新陈代谢。要满足生理平衡的需求,理想的人工抗氧化酶不仅要具备较高的活力,还需要能被很好地调控,从而智能地行使其功能。为了实现该目标,首先要根据传统的酶催化设计方法,构建出高效的活性中心,再利用多种刺激响应材料的属性,将对于光、温度、p H等外界刺激敏感的特性加入到人工酶中,就能赋予人工酶智能的性质。随着刺激响应型材料的蓬勃发展和广泛应用,大量具有智能性质的体系被发掘出来。如光敏感的偶氮苯变构行为、温敏聚合物的构象变化和超分子系统的组装等可逆过程都可以被用来控制酶的开关调控。而相对于合成分子,天然的别构蛋白质骨架不仅具有和天然酶相似的结构,还凭借较高的生物相容性、别构调控精度和适应性引起了科学家们的注意。借助于计算机辅助设计和相关理论手段,在非同源蛋白质上设计全新的催化位点已经成为了可能。因此,我们利用别构蛋白为骨架构建了高效的人工抗氧化酶,并借助于别构蛋白能够在一定条件下改变自身构象的特性,实现了对该人工抗氧化酶活力的调控。考虑到一些与细胞氧化水平紧密相关的生物信号,钙离子(Ca~(2+))和三磷酸腺苷(ATP)被选为人工抗氧化酶的调控分子。与之对应的,我们找到了恢复蛋白(Recoverin)和腺苷酸激酶(Adenylate Kinase,AKe),两种分别通过结合Ca~(2+)和ATP的别构蛋白作为骨架,成功构建出了Ca~(2+)响应型和ATP响应型智能人工抗氧化酶。这两种抗氧化酶不仅具有与天然酶接近的催化活力,还可以进行反复可逆的开关调控,并且在亚细胞水平上具有可控的生物学效应,为智能酶的研究和发展注入了新的活力。1.构建钙离子响应型智能人工抗氧化酶我们首先构建了Ca~(2+)响应型智能人工抗氧化酶。Ca~(2+)介导了从生物大分子氧化损伤到细胞凋亡的细胞信号通路,同时Ca~(2+)富集的线粒体等位置也是细胞氧化水平最严重的位置之一。为了在高浓度Ca~(2+)存在时发挥抗氧化酶的功能,Ca~(2+)结合蛋白Recoverin被选为人工酶的骨架。当Recoverin结合Ca~(2+)时,其处于一种相对松散的构象,N-terminal的豆蔻酰基团从Recoverin的内部被推出来,使得疏水空腔暴露在蛋白质表面;而当Recoverin不结合Ca~(2+)时,N-terminal的豆蔻酰基团会被包埋在保守的疏水空腔中,此时Recoveirn处于一种紧凑的构象。利用基因工程技术将天然GPx的催化中心Sec植入到128位之后,该催化位点会在Ca~(2+)存在时裸露在外,能够行使催化功能,而反之则被包埋在蛋白质内部,无法进行催化。根据这样的设计,能够建立酶活力的Ca~(2+)开关响应性质,并做到了反复多个循环的开关调控而无明显的活力衰减。进一步地,为了保证该人工酶的催化活力,我们结合天然GPx的催化机制,在催化中心的附近添加了有利于底物结合的精氨酸,成功地将催化活力提升了近六倍,达到与人血浆GPx同一数量级的活力。计算机模拟显示,对催化中心附近微环境的改进极大地提高了分子对接的形状互补性和蛋白质表面正电势,提升了酶-底物亲和力,最终大幅提升了催化活力。2.构建ATP响应型智能人工抗氧化酶其次,我们构建了ATP响应型智能人工抗氧化酶。在病理条件下的缺血再灌注过程中,严重的氧化损伤往往伴随着ATP浓度的大幅下降,因此ATP浓度与氧化损伤呈负相关,ATP浓度可以作为一种活性氧自由基水平的指标。AKe作为一种ATP敏感的蛋白质骨架,在不结合ATP时呈现一种开启状态的构象,其核心结构域裸露在表面。而当结合ATP时,AKe转变为一种关闭的状态,处于紧凑的构象。我们利用AKe上120号位点在不同构象下裸露程度不同的特性,在此处利用基因工程方法植入Sec催化中心,得到了一种可被体系中ATP抑制活力的人工抗氧化酶。值得注意的是,在ATP被同体系中的己糖激酶所消耗掉之后,该人工抗氧化酶仍可以恢复活力,并且能够反复循环调控多次。为了研究催化位点周围关键氨基酸对于稳定底物结合的作用,一系列定点突变被用于验证底物结合的相关机制,并通过实验发现在137位置引入精氨酸可以提高酶-底物亲和力,有利于催化活力的提高。最终,该ATP响应型人工抗氧化酶的催化活性也达到了与人血浆GPx相同数量级的活力水平,并以较高的灵敏度被ATP浓度所调控。3.智能人工抗氧化酶的生物学效应我们所构建的两种智能人工抗氧化酶能够以可调控的方式保护线粒体免受ROS损伤。在亚细胞水平下,我们利用Vc/Fe_(2+)自由基诱发体系模拟线粒体内的氧化损伤环境,并在该体系中加入上述的两种人工抗氧化酶,检验其保护线粒体的功能。在强烈的氧化压力下,线粒体会发生明显的膨胀现象,结构完整性下降并发生脂质过氧化反应。在520 nm波长下测线粒体的吸收值,如果该吸收值的下降说明体系浊度下降,线粒体膨胀度增加,线粒体逐渐失去完整性。线粒体中的不饱和脂肪酸被H2O2氧化成环氧化物,并经历一系列代谢过程最终生成丙二醛(MDA)。MDA与硫代巴比妥酸(TBA)在酸性加热条件下反应生成粉红色产物,该产物在532 nm处显示最大的吸收值。实验证明,两种人工酶都可以明显地抑制氧化损伤导致的线粒体膨胀和脂质过氧化,并且在调控分子存在与否的不同情况体现出了良好的开关效应。
[Abstract]:Reactive Oxygen Species (ROS), produced by oxygen breathing in cells, can cause damage to biological macromolecules in the body, resulting in a series of major diseases such as cataracts, Keshan disease, Alzheimer's disease, cardiogenic shock and atherosclerosis. Glutathione peroxidase (GPx) can be used as the bottom of the glutathione molecule (GSH). It uses its catalytic center selenocysteine (Sec) to catalyze the reduction of peroxides to protect biological macromolecules from ROS damage. In view of the excellent antioxidant effects of GPx, artificial analog GPx has become one of the hot spots in the field of enzyme mimics. Especially in the catalytic activity, the catalytic efficiency near the natural enzyme has been all the time. It is the goal of scientists. The GPx model of small molecules, to selenium antibody, semi synthetase, bioimprinted enzyme, and protein GPx enzyme model, a large number of highly active artificial GPx analogue enzymes are constructed. These results are of great significance for the prevention and control of disease, delay aging and other fields. The regulatory mechanism is also one of the important characteristics of natural enzymes,.ROS is not always harmful. Under normal physiological conditions, they play an important role in cell signal transduction. The homeostasis of organisms is maintained by a variety of mechanisms. If an effective anti oxidizing drug is taken simply, the metabolic system of the body will be lost. In order to meet the needs of physiological balance, the ideal artificial antioxidant enzyme should not only have high vitality, but also need to be well regulated to perform its function intelligently. In order to achieve this goal, first of all, it is necessary to build efficient active center based on the traditional enzyme catalytic design method. With the properties of a variety of stimuli responsive materials and the addition of the sensitive characteristics of light, temperature, and P H to the artificial enzyme, the intelligent properties of artificial enzymes can be given. With the vigorous development and extensive application of the stimuli responsive materials, a large number of systems with intelligent properties have been discovered. Such as the behavior of photoperiod sensitive azobenzene, Wen Min The reversible processes, such as conformation changes of polymers and assembly of supramolecular systems, can be used to control the switch regulation of enzymes. Compared with synthetic molecules, natural allosteric protein skeletons not only have similar structures with natural enzymes, but also have high biocompatibility. The precision and adaptability of the allosteric modulation and control have aroused the attention of scientists. With the help of computer aided design and related theoretical means, it is possible to design new catalytic sites on non homologous proteins. Therefore, we use the allosteric protein as the skeleton to construct a highly efficient artificial antioxidant enzyme, and by the aid of the allosteric protein can change the characteristics of its conformation under certain conditions and realize the artificial artificial protein. Regulation of antioxidant enzyme activity. Considering some biological signals closely related to the level of cell oxidation, calcium ions (Ca~ (2+)) and adenosine triphosphate (ATP) are selected as the regulators of artificial antioxidant enzymes. Corresponding to this, we found the recovery protein (Recoverin) and adenylate kinase (Adenylate Kinase, AKe), two by combining Ca~ (Adenylate Kinase), respectively. 2+) and ATP's allosteric protein as the skeleton, successfully constructed the Ca~ (2+) responsive and ATP responsive intelligent artificial antioxidant enzymes. These two antioxidant enzymes not only have catalytic activity close to natural enzymes, but also have reversible and reversible switching regulation, and have controllable biological effects on the subcellular water level, which are the Research of intelligent enzymes. Research and development infuse new vitality.1. construction calcium responsive intelligent artificial antioxidant enzyme. We first constructed Ca~ (2+) responsive intelligent artificial antioxidant enzyme.Ca~ (2+) mediated cell signaling pathway from biological macromolecular oxidation to apoptosis, and Ca~ (2+) enriched mitochondria are also the most severe oxidative level of cells. One of the heavy positions. In order to function as an antioxidant enzyme in the presence of high concentration Ca~ (2+), the Ca~ (2+) binding protein Recoverin is selected as the skeleton of an artificial enzyme. When Recoverin combines Ca~ (2+), it is in a relatively loose conformation, and N-terminal's myristic group is pushed out of the Recoverin, causing the hydrophobic cavity to be exposed to the protein. When Recoverin does not combine with Ca~ (2+), the cardamomoyl group of N-terminal is embedded in the conservative hydrophobic cavity, when Recoveirn is in a compact conformation. After the implantation of the catalytic center Sec of natural GPx to 128 sites by genetic engineering, the catalytic site will be exposed in the presence of Ca~ (2+) and can exercise the urge. In this design, the enzyme activity Ca~ (2+) switch responds to the nature of the enzyme activity, and the multiple cycles of the switch are regulated without significant energy decay. Further, in order to ensure the catalytic activity of the artificial enzyme, we combine the catalysis of natural GPx. The mechanism, in the vicinity of the catalytic center, added the arginine which is beneficial to the substrate binding, successfully promoted the catalytic activity by nearly six times and reached the same order of magnitude as the human plasma GPx. The computer simulation showed that the improvement of the shape complementarity of the sub docking and the positive potential of protein surface greatly improved the micro environment near the catalytic center. The enzyme substrate affinity was enhanced, and the catalytic activity of.2. was ultimately greatly enhanced by the construction of a ATP responsive intelligent artificial antioxidant enzyme. We constructed a ATP responsive intelligent artificial antioxidant enzyme. In the pathological condition, severe oxidative damage often accompanied by a significant decrease in the concentration of ATP, thus ATP concentration and oxidation The damage is negatively correlated, and ATP concentration can be used as an indicator of active oxygen free radical level.AKe as a ATP sensitive protein skeleton, which presents a state of open conformation when it does not combine with ATP, and its core domain is exposed to the surface. When combined with ATP, AKe is turned into a closed state and is in a compact conformation. We use AKe. The upper 120 loci were exposed to different conformations in different conformations, where a genetic engineering method was used to implant the Sec catalytic center, and an artificial antioxidant enzyme that could be inhibited by ATP in the system was obtained. It is worth noting that the artificial antioxidant can still rejuvenate after ATP is consumed by hexose kinase in the same system. In order to study the effect of key amino acids around the catalytic site on the stabilization of substrate binding, a series of site directed mutagenesis was used to verify the mechanism of substrate binding. It was found that the introduction of arginine in the 137 position could improve the affinity of the enzyme base and improve the catalytic activity. Finally, The catalytic activity of the ATP responsive artificial antioxidant enzyme also reached the same magnitude of activity as human plasma GPx, and the biological effects of.3. intelligent artificial antioxidase regulated by ATP concentration were high sensitivity. The two kinds of intelligent artificial antioxidant enzymes constructed by us could protect mitochondria from ROS damage in a controllable way. At the subcellular level, we used the Vc/Fe_ (2+) free radical induced system to simulate the oxidative damage environment in the mitochondria, and we added two kinds of artificial antioxidant enzymes in the system to test the function of mitochondria protection. Lipid peroxidation. The absorption values of mitochondria were measured at 520 nm wavelengths. If the decrease of the absorption value indicates that the turbidity of the system decreases, the expansion of mitochondria is increased and the mitochondria gradually lose integrity. The unsaturated fatty acids in mitochondria are oxidized to epoxides by H2O2, and a series of metabolic processes eventually produce malondialdehyde (MDA).MDA and The reaction of thiobarbituric acid (TBA) produces pink products under the condition of acid heating. The product shows the maximum absorption value at 532 nm. Experiments show that the two kinds of artificial enzymes can obviously inhibit the mitochondrial dilation and lipid peroxidation caused by oxidative damage, and are good in regulating the presence or not of the molecules. Switch effect.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：O629.8

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