丙酮酸激酶在活化离子和底物诱导下的构象变化机理研究

发布时间：2018-03-26 20:12

本文选题：丙酮酸激酶　切入点：构象变化　出处：《复旦大学》2010年硕士论文

【摘要】： 丙酮酸激酶(pyruvate kinase, PK)是糖酵解途径中的一个关键酶。在糖酵解途径中,PK在活化离子K+和Mg2+存在条件下,催化磷酸稀醇式丙酮酸(PEP)和二磷酸腺苷(ADP)转变为丙酮酸和三磷酸腺苷(ATP)。PK活性部位的结构表明,结构域B伸出到溶液中与结构域A形成一个口袋,活性部位就处在同一个亚基的结构域A和结构域B之间的口袋中,结构域B通过一个易弯曲的交联区域与结构域A相连。结构域B的旋转使得结构域A和结构域B之间的口袋“张开”代表了PK的非活性状态,口袋的“闭合”代表了PK的活性状态。结构域B具有高度的活动性,在两种构象中有40°的角度变化,而结构域C在“张开”和“闭合”的构象中都处在同样的位置。PK的活化离子-底物复合物的构象不同于自由酶的构象。文献中紫外光谱和荧光光谱研究表明在活化离子存在下或者温度降低时,蛋白的某些生色团由非水性环境变为水性环境。底物ADP在活化离子存在下并不干扰蛋白的紫外光谱。PEP的结合使得PK的结构更加紧密,也更加对称。相反,抑制剂苯丙氨酸(Phe)使得PK的结构更加松散。活化离子K+和Mg2+、底物ADP和PEP、抑制剂Phe对PK的二级结构都没有引起显著的变化。本文主要应用荧光淬灭技术研究了活化离子K+和Mg2+、底物ADP和PEP、抑制剂Phe、温度、盐酸胍对PK构象变化的影响,并尝试用等温滴定量热技术(ITC)进行PK与活化离子、底物之间的热动力学研究,以期探讨PK在诸多因素诱导下的构象变化规律及其与热动力学函数之间的关系。本研究得到了以下结论： 1、活化离子K+和Mg2+、底物ADP和PEP、抑制剂Phe等对丙酮酸激酶的结构变化影响均很微弱,但能引起丙酮酸激酶结构域的移动和构象变化。在活化离子Mg2+或者Mg2+和K+共同作用下,丙酮酸激酶的活性部位更加暴露,处于更加亲水的环境。底物ADP对于丙酮酸激酶活性部位暴露程度的变化几乎没有作用,底物PEP的结合或者PEP和ADP共同的作用能明显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,还能显著地增大色氨酸残基的暴露程度。 2、温度在10-30℃之间变化时,丙酮酸激酶的整体二级结构没有监测到变化,但能够引起丙酮酸激酶活性部位的构象变化。活性部位的暴露程度与温度之间有反相关性,温度越低活性部位暴露程度越高。 3、PK在变性剂盐酸胍(GdnHCl)作用下的去折叠规律：当盐酸胍浓度在0.5M时,丙酮酸激酶解离成一个松散且失活的四聚体；当盐酸胍浓度达到1.5M时,丙酮酸激酶解离成扩大的二聚体,此时已经有部分二级结构丢失；当盐酸胍浓度进一步增大到2.5M时,丙酮酸激酶解离成完全无序的单体。但活化离子和底物能够部分抑制由于盐酸胍引起的丙酮酸激酶解离。本文还对蛋白磷酸化酶(Calcineurin, CN)在钙离子和钙调节蛋白(CaM)诱导下的构象变化机理进行了研究。CN是一个依赖于钙离子/钙调节蛋白(Ca2+/CaM)的丝氨酸/苏氨酸磷酸化酶,参与大量的细胞内信号的调节。CN是由A,B两个亚基组成的异二聚体蛋白酶：A亚基(CNA,61-kDa)是催化亚基,主要起催化作用；B亚基(CNB,19-kDa)是调节亚基,对酶的活性起着调节的作用。本论文通过丙烯酰胺荧光淬灭技术研究了CN在钙离子和钙调节蛋白诱导下的构象变化机理,得出如下结论：CN自我调节结构域(CNRR)通过封闭CN的活性部位而抑制CN的活性,而CaM的结合使得活性部位暴露出来；Ca2+结合到CNB上能够激活CaM结合到CNA的调节区域,然后在不需要CNB的情况下,CaM能够独自引起自我调节结构域发生构象变化,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.

【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R341

【引证文献】