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

【作者】 欧艳；

【导师】 余绍宁；

【作者基本信息】 复旦大学 ， 化学生物学， 2010， 硕士

【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。更多还原

【Abstract】 Pyruvate kinase (PK) is a key regulatory glycolytic enzyme. PK catalyzes the physiological phosphorylation of ADP by PEP. The active-site structure of PK showed that domain B of PK protrudes into the solvent and forms a cleft with domain A. Domain B is attached to domain A by what appears to be a flexible hinge region. The active site lies in the pocket between domains A and B of the same subunit. The inactive state can be represented by a rotation of the domain B in opening of the cleft between the B and A domains, and the active state can be represented by closing of the cleft. The domain B is highly mobile and differs in 40°in the two conformers, whereas the domain C remains in the same position in the "open" and "closed" conformations. The enzyme-metal-substrate complex has a conformation which is different from that of free enzyme. The UV and fluorescence specrtrum studies reveal that the solvating environment of certain protein chromophores is in a non-aqueous environment at high temperature or in the absence of cations, and in an aqueous environment at low temperature or in the presence of cations. The substrate, ADP, didn’t perturb the UV spectrum of the protein in the presence of activating cations. Binding of PEP induces PK to assume a more compact and symmetric structure. In contrast, Phe loosens the protein structure into an inactive or less active state. The binding of activating cations and/or substrates or the inhibitor retains the relative distribution of the secondary structures.In this work, the conformational change of PK induced by its activating cations K+/Mg2+, substrates ADP/PEP, and inhibitor Phe, have been studied by using fluorescence acrylamide quenching. Isothermal titration calorimetry (ITC) was used to address the thermodynamic properties of the reaction between PK and activating cations and/or substrates. The results are as follows:1. There is no significant change in the secondary structure in PK induced by its activating matels K+/Mg2+, substrates ADP/PEP, inhibitor Phe. However, it involves domain movements and conformational changes. The active site of PK was brought into an aqueous environment by interactions of Mg2+ or K+/Mg2+. ADP has little contribution on the solvent accessibility of tryptophan residues. Binding of PEP or PEP/ADP elicits a decrease in accessibility of PK active site. Phe can inhibit the activity of PK and in contract substrate PEP, increase the solvent accessibility of PK tryptophan residues.2. When enzyme is heated from 10℃to 30℃, there is also no significant change in the secondary structure. However, temperature would change the conformation of PK active site, the solvent accessibility of active site is inversely related to temperature.3. As a homo-tetramer, PK can be dissociated into a less compact and in-active tetramer in the presence of 0.5M GdnHCl. In the presence of 1.5M GdnHCl, PK was dissociated into dimmers dimer with a partial loss of the secondary structure. PK is a disordered monomer in the presence of 2.5M GdnHCl. Noteworthy, activating cations and substrates can partially reverse the conformational change induced by GdnHCl.The conformational changes of Calcineurin (CN) induced by Ca2+/CaM binding was also studied in this work. CN is a calcium/CaM-dependent Ser/Thr protein phosphatase and plays a critical role in the coupling of Ca2+signals to cellular responses. CN is a heterodimeric enzyme consisting of a 61-kDa subunit (CNA) with catalytic activity and the binding sites for Ca2+/CaM and a 19-kDa subunit (CNB). The conformational change mechanism was studied by fluorescence acrylamide quenching. The results are:the isolated regulatory region (CNRR) inhibits CN activity by occluding the catalytic site and that CaM binding exposes the catalytic site. The binding of Ca2+ to CNB enables CaM binding to the CNA regulatory region, and CaM binding then instructs an activating conformational change of the regulatory region that does not depend further on CNB.更多还原