Functional energetic landscape in the allosteric regulation of muscle pyruvate kinase. 2. Fluorescence study.
Functional energetic landscape in the allosteric regulation of muscle pyruvate kinase. 2. Fluorescence study.
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DOI：
10.1021/bi900280u
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发表时间：
2009-10-13
期刊：
影响因子：
2.9
通讯作者：
Lee, J. Ching
中科院分区：
文献类型：
--
作者：
Herman, Petr;Lee, J. Ching
用等温滴定量热法(ITC)表征了兔肌丙酮酸激酶(RMPK)变构调节机制的能量图景。发现了四个新的见解：1.ADP表现出双重属性。根据温度的不同，ADP可以通过将酶切换到R状态或T状态来调节RMPK的活性。2.配体与RMPK的结合是状态依赖的假设只适用于PEP，而不适用于Phe/ADP。3.pH对RMPK调节行为的影响部分是由于质子释放或吸收的复杂模式，这些模式与控制酶活性的多个连锁平衡有关。4.R-↔-T平衡伴随着显著的ΔCP，使其在生理条件下对温度最为敏感。为了严格检验从国贸中心数据得出的结论的有效性，在这项研究中，使用了一种荧光方法，尽管是间接的，但跟踪连续的结构扰动。在4°C到45°C的温度范围内测量了RMPK在无底物和底物磷酸烯醇式丙酮酸(PEP)和ADP存在时的本征Trp荧光，以及变构抑制剂Phe。为了进行数据分析，ITC实验对荧光数据进行了补充，以获得扩展的数据集，从而可以更完整地描述RMPK的调控机制。通过对ITC和荧光数据集的全球分析，得出了21个热力学参数，以定义参与调节RMPK变构行为的链接相互作用网络。在这项研究中，对超过1600个实验点的27条独立曲线进行了全局分析。因此，一致的结果不仅证实了从ITC数据得出的结论，而且还证实了RMPK活性和非活性状态之间的转换以及ADP和Phe结合之间的拮抗作用的结构信息。后者揭示了ADP在RMPK变构调节中的新作用。
The energetic landscape of the allosteric regulatory mechanism of rabbit muscle pyruvate kinase (RMPK) was characterized by isothermal titration calorimetry (ITC). Four novel insights were uncovered 1. ADP exhibits a dual property. Depending on the temperature, ADP can regulate RMPK activity by switching the enzyme to either the R- or T-state. 2. The assumption that ligand binding to RMPK is state dependent is only correct for PEP but not Phe/ADP. 3. The pH effect on the regulatory behavior of RMPK is partly due to the complex pattern of proton release or absorption linked to the multiple linked equilibria which govern the activity of the enzyme. 4. The R↔T equilibrium is accompanied by a significant ΔCp rendering RMPK most sensitive to temperature under physiological conditions. In order to rigorously test the validity of conclusions derived from the ITC data, in this study a fluorescence approach, albeit indirect, that tracks continuous structural perturbations was employed. Intrinsic Trp fluorescence of RMPK in the absence and in the presence of substrates phosphoenolpyruvate (PEP) and ADP, and the allosteric inhibitor Phe was measured in the temperature range between 4°C and 45°C. For data analysis the fluorescence data were complemented by ITC experiments to obtain extended data set allowing more complete characterization of the RMPK regulatory mechanism. Twenty-one thermodynamic parameters were derived to define the network of linked interactions involved in regulating the allosteric behavior of RMPK through global analysis of the ITC and fluorescent data sets. In this study 27 independent curves with more than 1600 experimental points were globally analyzed. Consequently, the consensus results not only substantiate the conclusions derived from the ITC data but also structural information characterizing the transition between the active and the inactive state of RMPK and the antagonism between ADP and Phe binding. The latter observation reveals a novel role for ADP in the allosteric regulation of RMPK.
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