本文選題：谷胱甘肽過氧化物酶 + 別構(gòu)蛋白��； 參考：《吉林大學(xué)》2017年博士論文

【摘要】：細(xì)胞有氧呼吸產(chǎn)生的活性氧自由基(Reactive Oxygen Species,ROS)會(huì)引起機(jī)體內(nèi)生物大分子的損傷,進(jìn)而導(dǎo)致白內(nèi)障、克山病、阿茲海默癥、心源性休克以及動(dòng)脈粥樣硬化等一系列重大疾病。谷胱甘肽過氧化物酶(GPx)能夠以谷胱甘肽分子(GSH)為底物,利用其催化中心硒代半胱氨酸(Sec)催化過氧化物的還原,從而保護(hù)生物大分子免受ROS的損傷。鑒于GPx具有出色的抗氧化效果,人工模擬GPx成為了仿酶領(lǐng)域的熱點(diǎn)之一。尤其是在催化反應(yīng)活力方面,接近于天然酶的催化效率一直以來都是科學(xué)家們追求的目標(biāo)。從小分子的GPx模型,到含硒抗體、半合成酶、生物印跡酶以及蛋白質(zhì)GPx酶模型,大量具有高活力的人工GPx模擬酶被構(gòu)建出來。這些成果對(duì)于疾病防治、延緩衰老等領(lǐng)域有著重要意義。除了具有極高的催化效率,嚴(yán)格的調(diào)控機(jī)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?xiàng)l件下,它們?cè)诩?xì)胞信號(hào)轉(zhuǎn)導(dǎo)方面起著重要作用。生物體內(nèi)的穩(wěn)態(tài)是由多種機(jī)制共同維持的,如果單純地服用高效的抗氧化藥物,必將導(dǎo)致機(jī)體的代謝系統(tǒng)失調(diào),反而不利于正常的新陳代謝。要滿足生理平衡的需求,理想的人工抗氧化酶不僅要具備較高的活力,還需要能被很好地調(diào)控,從而智能地行使其功能。為了實(shí)現(xiàn)該目標(biāo),首先要根據(jù)傳統(tǒng)的酶催化設(shè)計(jì)方法,構(gòu)建出高效的活性中心,再利用多種刺激響應(yīng)材料的屬性,將對(duì)于光、溫度、p H等外界刺激敏感的特性加入到人工酶中,就能賦予人工酶智能的性質(zhì)。隨著刺激響應(yīng)型材料的蓬勃發(fā)展和廣泛應(yīng)用,大量具有智能性質(zhì)的體系被發(fā)掘出來。如光敏感的偶氮苯變構(gòu)行為、溫敏聚合物的構(gòu)象變化和超分子系統(tǒng)的組裝等可逆過程都可以被用來控制酶的開關(guān)調(diào)控。而相對(duì)于合成分子,天然的別構(gòu)蛋白質(zhì)骨架不僅具有和天然酶相似的結(jié)構(gòu),還憑借較高的生物相容性、別構(gòu)調(diào)控精度和適應(yīng)性引起了科學(xué)家們的注意。借助于計(jì)算機(jī)輔助設(shè)計(jì)和相關(guān)理論手段,在非同源蛋白質(zhì)上設(shè)計(jì)全新的催化位點(diǎn)已經(jīng)成為了可能。因此,我們利用別構(gòu)蛋白為骨架構(gòu)建了高效的人工抗氧化酶,并借助于別構(gòu)蛋白能夠在一定條件下改變自身構(gòu)象的特性,實(shí)現(xiàn)了對(duì)該人工抗氧化酶活力的調(diào)控�？紤]到一些與細(xì)胞氧化水平緊密相關(guān)的生物信號(hào),鈣離子(Ca~(2+))和三磷酸腺苷(ATP)被選為人工抗氧化酶的調(diào)控分子。與之對(duì)應(yīng)的,我們找到了恢復(fù)蛋白(Recoverin)和腺苷酸激酶(Adenylate Kinase,AKe),兩種分別通過結(jié)合Ca~(2+)和ATP的別構(gòu)蛋白作為骨架,成功構(gòu)建出了Ca~(2+)響應(yīng)型和ATP響應(yīng)型智能人工抗氧化酶。這兩種抗氧化酶不僅具有與天然酶接近的催化活力,還可以進(jìn)行反復(fù)可逆的開關(guān)調(diào)控,并且在亞細(xì)胞水平上具有可控的生物學(xué)效應(yīng),為智能酶的研究和發(fā)展注入了新的活力。1.構(gòu)建鈣離子響應(yīng)型智能人工抗氧化酶我們首先構(gòu)建了Ca~(2+)響應(yīng)型智能人工抗氧化酶。Ca~(2+)介導(dǎo)了從生物大分子氧化損傷到細(xì)胞凋亡的細(xì)胞信號(hào)通路,同時(shí)Ca~(2+)富集的線粒體等位置也是細(xì)胞氧化水平最嚴(yán)重的位置之一。為了在高濃度Ca~(2+)存在時(shí)發(fā)揮抗氧化酶的功能,Ca~(2+)結(jié)合蛋白R(shí)ecoverin被選為人工酶的骨架。當(dāng)Recoverin結(jié)合Ca~(2+)時(shí),其處于一種相對(duì)松散的構(gòu)象,N-terminal的豆蔻�；鶊F(tuán)從Recoverin的內(nèi)部被推出來,使得疏水空腔暴露在蛋白質(zhì)表面;而當(dāng)Recoverin不結(jié)合Ca~(2+)時(shí),N-terminal的豆蔻�；鶊F(tuán)會(huì)被包埋在保守的疏水空腔中,此時(shí)Recoveirn處于一種緊湊的構(gòu)象。利用基因工程技術(shù)將天然GPx的催化中心Sec植入到128位之后,該催化位點(diǎn)會(huì)在Ca~(2+)存在時(shí)裸露在外,能夠行使催化功能,而反之則被包埋在蛋白質(zhì)內(nèi)部,無法進(jìn)行催化。根據(jù)這樣的設(shè)計(jì),能夠建立酶活力的Ca~(2+)開關(guān)響應(yīng)性質(zhì),并做到了反復(fù)多個(gè)循環(huán)的開關(guān)調(diào)控而無明顯的活力衰減。進(jìn)一步地,為了保證該人工酶的催化活力,我們結(jié)合天然GPx的催化機(jī)制,在催化中心的附近添加了有利于底物結(jié)合的精氨酸,成功地將催化活力提升了近六倍,達(dá)到與人血漿GPx同一數(shù)量級(jí)的活力。計(jì)算機(jī)模擬顯示,對(duì)催化中心附近微環(huán)境的改進(jìn)極大地提高了分子對(duì)接的形狀互補(bǔ)性和蛋白質(zhì)表面正電勢(shì),提升了酶-底物親和力,最終大幅提升了催化活力。2.構(gòu)建ATP響應(yīng)型智能人工抗氧化酶其次,我們構(gòu)建了ATP響應(yīng)型智能人工抗氧化酶。在病理?xiàng)l件下的缺血再灌注過程中,嚴(yán)重的氧化損傷往往伴隨著ATP濃度的大幅下降,因此ATP濃度與氧化損傷呈負(fù)相關(guān),ATP濃度可以作為一種活性氧自由基水平的指標(biāo)。AKe作為一種ATP敏感的蛋白質(zhì)骨架,在不結(jié)合ATP時(shí)呈現(xiàn)一種開啟狀態(tài)的構(gòu)象,其核心結(jié)構(gòu)域裸露在表面。而當(dāng)結(jié)合ATP時(shí),AKe轉(zhuǎn)變?yōu)橐环N關(guān)閉的狀態(tài),處于緊湊的構(gòu)象。我們利用AKe上120號(hào)位點(diǎn)在不同構(gòu)象下裸露程度不同的特性,在此處利用基因工程方法植入Sec催化中心,得到了一種可被體系中ATP抑制活力的人工抗氧化酶。值得注意的是,在ATP被同體系中的己糖激酶所消耗掉之后,該人工抗氧化酶仍可以恢復(fù)活力,并且能夠反復(fù)循環(huán)調(diào)控多次。為了研究催化位點(diǎn)周圍關(guān)鍵氨基酸對(duì)于穩(wěn)定底物結(jié)合的作用,一系列定點(diǎn)突變被用于驗(yàn)證底物結(jié)合的相關(guān)機(jī)制,并通過實(shí)驗(yàn)發(fā)現(xiàn)在137位置引入精氨酸可以提高酶-底物親和力,有利于催化活力的提高。最終,該ATP響應(yīng)型人工抗氧化酶的催化活性也達(dá)到了與人血漿GPx相同數(shù)量級(jí)的活力水平,并以較高的靈敏度被ATP濃度所調(diào)控。3.智能人工抗氧化酶的生物學(xué)效應(yīng)我們所構(gòu)建的兩種智能人工抗氧化酶能夠以可調(diào)控的方式保護(hù)線粒體免受ROS損傷。在亞細(xì)胞水平下,我們利用Vc/Fe_(2+)自由基誘發(fā)體系模擬線粒體內(nèi)的氧化損傷環(huán)境,并在該體系中加入上述的兩種人工抗氧化酶,檢驗(yàn)其保護(hù)線粒體的功能。在強(qiáng)烈的氧化壓力下,線粒體會(huì)發(fā)生明顯的膨脹現(xiàn)象,結(jié)構(gòu)完整性下降并發(fā)生脂質(zhì)過氧化反應(yīng)。在520 nm波長(zhǎng)下測(cè)線粒體的吸收值,如果該吸收值的下降說明體系濁度下降,線粒體膨脹度增加,線粒體逐漸失去完整性。線粒體中的不飽和脂肪酸被H2O2氧化成環(huán)氧化物,并經(jīng)歷一系列代謝過程最終生成丙二醛(MDA)。MDA與硫代巴比妥酸(TBA)在酸性加熱條件下反應(yīng)生成粉紅色產(chǎn)物,該產(chǎn)物在532 nm處顯示最大的吸收值。實(shí)驗(yàn)證明,兩種人工酶都可以明顯地抑制氧化損傷導(dǎo)致的線粒體膨脹和脂質(zhì)過氧化,并且在調(diào)控分子存在與否的不同情況體現(xiàn)出了良好的開關(guān)效應(yīng)。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：O629.8

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