本文選題：丙酮酸激酶　切入點(diǎn)：構(gòu)象變化　出處：《復(fù)旦大學(xué)》2010年碩士論文

【摘要】： 丙酮酸激酶(pyruvate kinase, PK)是糖酵解途徑中的一個(gè)關(guān)鍵酶。在糖酵解途徑中,PK在活化離子K+和Mg2+存在條件下,催化磷酸稀醇式丙酮酸(PEP)和二磷酸腺苷(ADP)轉(zhuǎn)變?yōu)楸岷腿姿嵯佘?ATP)。PK活性部位的結(jié)構(gòu)表明,結(jié)構(gòu)域B伸出到溶液中與結(jié)構(gòu)域A形成一個(gè)口袋,活性部位就處在同一個(gè)亞基的結(jié)構(gòu)域A和結(jié)構(gòu)域B之間的口袋中,結(jié)構(gòu)域B通過一個(gè)易彎曲的交聯(lián)區(qū)域與結(jié)構(gòu)域A相連。結(jié)構(gòu)域B的旋轉(zhuǎn)使得結(jié)構(gòu)域A和結(jié)構(gòu)域B之間的口袋“張開”代表了PK的非活性狀態(tài),口袋的“閉合”代表了PK的活性狀態(tài)。結(jié)構(gòu)域B具有高度的活動(dòng)性,在兩種構(gòu)象中有40°的角度變化,而結(jié)構(gòu)域C在“張開”和“閉合”的構(gòu)象中都處在同樣的位置。PK的活化離子-底物復(fù)合物的構(gòu)象不同于自由酶的構(gòu)象。文獻(xiàn)中紫外光譜和熒光光譜研究表明在活化離子存在下或者溫度降低時(shí),蛋白的某些生色團(tuán)由非水性環(huán)境變?yōu)樗原h(huán)境。底物ADP在活化離子存在下并不干擾蛋白的紫外光譜。PEP的結(jié)合使得PK的結(jié)構(gòu)更加緊密,也更加對(duì)稱。相反,抑制劑苯丙氨酸(Phe)使得PK的結(jié)構(gòu)更加松散�；罨x子K+和Mg2+、底物ADP和PEP、抑制劑Phe對(duì)PK的二級(jí)結(jié)構(gòu)都沒有引起顯著的變化。 本文主要應(yīng)用熒光淬滅技術(shù)研究了活化離子K+和Mg2+、底物ADP和PEP、抑制劑Phe、溫度、鹽酸胍對(duì)PK構(gòu)象變化的影響,并嘗試用等溫滴定量熱技術(shù)(ITC)進(jìn)行PK與活化離子、底物之間的熱動(dòng)力學(xué)研究,以期探討PK在諸多因素誘導(dǎo)下的構(gòu)象變化規(guī)律及其與熱動(dòng)力學(xué)函數(shù)之間的關(guān)系。本研究得到了以下結(jié)論： 1、活化離子K+和Mg2+、底物ADP和PEP、抑制劑Phe等對(duì)丙酮酸激酶的結(jié)構(gòu)變化影響均很微弱,但能引起丙酮酸激酶結(jié)構(gòu)域的移動(dòng)和構(gòu)象變化。在活化離子Mg2+或者M(jìn)g2+和K+共同作用下,丙酮酸激酶的活性部位更加暴露,處于更加親水的環(huán)境。底物ADP對(duì)于丙酮酸激酶活性部位暴露程度的變化幾乎沒有作用,底物PEP的結(jié)合或者PEP和ADP共同的作用能明顯降低丙酮酸激酶活性部位的暴露程度。Phe能抑制丙酮酸激酶的活性,還能顯著地增大色氨酸殘基的暴露程度。 2、溫度在10-30℃之間變化時(shí),丙酮酸激酶的整體二級(jí)結(jié)構(gòu)沒有監(jiān)測(cè)到變化,但能夠引起丙酮酸激酶活性部位的構(gòu)象變化。活性部位的暴露程度與溫度之間有反相關(guān)性,溫度越低活性部位暴露程度越高。 3、PK在變性劑鹽酸胍(GdnHCl)作用下的去折疊規(guī)律：當(dāng)鹽酸胍濃度在0.5M時(shí),丙酮酸激酶解離成一個(gè)松散且失活的四聚體；當(dāng)鹽酸胍濃度達(dá)到1.5M時(shí),丙酮酸激酶解離成擴(kuò)大的二聚體,此時(shí)已經(jīng)有部分二級(jí)結(jié)構(gòu)丟失；當(dāng)鹽酸胍濃度進(jìn)一步增大到2.5M時(shí),丙酮酸激酶解離成完全無序的單體。但活化離子和底物能夠部分抑制由于鹽酸胍引起的丙酮酸激酶解離。 本文還對(duì)蛋白磷酸化酶(Calcineurin, CN)在鈣離子和鈣調(diào)節(jié)蛋白(CaM)誘導(dǎo)下的構(gòu)象變化機(jī)理進(jìn)行了研究。CN是一個(gè)依賴于鈣離子/鈣調(diào)節(jié)蛋白(Ca2+/CaM)的絲氨酸/蘇氨酸磷酸化酶,參與大量的細(xì)胞內(nèi)信號(hào)的調(diào)節(jié)。CN是由A,B兩個(gè)亞基組成的異二聚體蛋白酶：A亞基(CNA,61-kDa)是催化亞基,主要起催化作用；B亞基(CNB,19-kDa)是調(diào)節(jié)亞基,對(duì)酶的活性起著調(diào)節(jié)的作用。本論文通過丙烯酰胺熒光淬滅技術(shù)研究了CN在鈣離子和鈣調(diào)節(jié)蛋白誘導(dǎo)下的構(gòu)象變化機(jī)理,得出如下結(jié)論：CN自我調(diào)節(jié)結(jié)構(gòu)域(CNRR)通過封閉CN的活性部位而抑制CN的活性,而CaM的結(jié)合使得活性部位暴露出來；Ca2+結(jié)合到CNB上能夠激活CaM結(jié)合到CNA的調(diào)節(jié)區(qū)域,然后在不需要CNB的情況下,CaM能夠獨(dú)自引起自我調(diào)節(jié)結(jié)構(gòu)域發(fā)生構(gòu)象變化,使CN得以激活。
[Abstract]:Pyruvate kinase (pyruvate kinase PK) is a key enzyme in the glycolytic pathway. In the glycolytic pathway, activation of K+ and PK in the presence of Mg2+ ion, catalytic phosphate diluted alcohol type pyruvate (PEP) and two AMP (ADP) into pyruvate and ATP (ATP) showed that the structure of the active site of.PK, B domain is extended to the solution and the A domain to form a pocket between domains A and B active site lies in the same subunit pocket, B domain of a flexible crosslinking region and A domain connected by rotation of the state. The activity between domains A and B pocket "open" on behalf of the PK domain of B, pocket "closed" on behalf of the active state of PK. B domain has the activity of height, change an angle of 40 degrees in two conformations, and C domain in "Zhang opening and closing" The conformation in the same conformation position.PK activated ion substrate complex conformation is different from that of free enzyme. The ultraviolet spectrum and fluorescence spectrum of the literature shows that in the presence of activating cations or at low temperature of certain protein chromophore by non aqueous environment into the water environment. Combined with UV spectra of.PEP substrate ADP does not interfere with protein activation in the presence of PK ions makes the structure more closely, more symmetrical. On the contrary, inhibitors of phenylalanine (Phe) makes the PK structure moreloose. Activation of ion K+ and Mg2+ substrate ADP and PEP, inhibit the two level structure of PK agent Phe did not cause significant change.
This paper mainly studies the application of fluorescence quenching technology of activated ion K+ and Mg2+, ADP and PEP substrates, inhibitors of Phe, temperature, effect of guanidine hydrochloride on the conformational change of PK, and using isothermal titration calorimetry (ITC) and PK activation ion, thermokinetic research between substrates, to explore the relationship between conformation changes in many factors induced by PK and thermodynamic function in this study obtained the following conclusions:
1, the activation of ion K+ and Mg2+, ADP and PEP substrates, inhibitors Phe structure change on pyruvate kinase effects are very weak, but can cause movement and conformational changes of pyruvate kinase domain. In the activation of ion Mg2+ or Mg2+ and K+ together, the activity of pyruvate kinase in a more exposed, more hydrophilic the environment. For pyruvate kinase active site substrate ADP exposure changes almost no effect, the substrate binding of PEP or PEP and ADP common function can significantly reduce the exposure extent of the active site of pyruvate kinase.Phe inhibits pyruvate kinase activity, but also significantly increases the exposure of tryptophan residues.
2, temperature at 10-30 DEG C when the whole two level structure of pyruvate kinase did not monitor changes, but can cause conformational changes in the kinase active site of pyruvate. The anti correlation between exposure and the temperature of the active site, the lower the temperature the higher the degree of exposure to the active site.
In 3, PK in denaturant guanidine hydrochloride (GdnHCl) under the action of the unfolding rule: when the concentration of guanidine hydrochloride in 0.5M, pyruvate kinase dissociation into a loose and four dimer inactivation; when guanidine hydrochloride concentration reached 1.5M, pyruvate kinase is dissociated into expanded two dimers, this time has been lost the two stage structure; when the guanidine concentration further increased to 2.5M, pyruvate kinase completely dissociated into monomers randomly. But activation of ion and substrate partially inhibited due to conformational change induced by GdnHCl.
The protein phosphorylase (Calcineurin, CN) in the regulation of protein calcium and calcium (CaM) induced conformational change mechanism is studied under.CN is a dependent on calcium / calmodulin (Ca2+/CaM) serine / threonine phosphorylase,.CN can regulate numerous intracellular signal by A, ISO the two mer protease B two subunits: A subunits (CNA, 61-kDa) is the catalytic subunit of the main catalytic subunit; B (CNB, 19-kDa) is the regulatory subunit, regulates the activity of enzyme. This paper research the CN regulation mechanism of protein induced conformational change in the presence of calcium and calcium by acrylamide fluorescent quenching technology, draw the following conclusions: CN self regulatory domain (CNRR) inhibition of CN by blocking CN and active site activity, and CaM combined with the active site exposed; Ca2+ binding to CNB can activate CaM. When it fits to the regulating area of CNA, and without the need for CNB, CaM can cause the conformation changes of the self regulating domain alone, enabling the CN to be activated.

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R341

【引證文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 呂凌;基于蛋白質(zhì)組學(xué)的脾虛大鼠脾失健運(yùn)機(jī)理的實(shí)驗(yàn)研究[D];遼寧中醫(yī)藥大學(xué);2012年

相關(guān)碩士學(xué)位論文 前1條

1 肖玲;松江鱸（Trachidermus fasciatus）六種糖代謝酶的基因克隆與序列分析[D];山東大學(xué);2011年

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本文編號(hào)：1669412