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首页> 外文期刊>Journal of Molecular Biology >Structural tightening and interdomain communication in the catalytic cycle of phosphoglycerate kinase.
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Structural tightening and interdomain communication in the catalytic cycle of phosphoglycerate kinase.

机译：磷酸甘油酸激酶催化循环中的结构收紧和域间通讯。

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摘要

Changes in amide-NH chemical shift and hydrogen exchange rates as phosphoglycerate kinase progresses through its catalytic cycle have been measured to assess whether they correlate with changes in hydrogen bonding within the protein. Four representative states were compared: the free enzyme, a product complex containing 3-phosphoglyceric acid (3PG), a substrate complex containing ADP and a transition-state analogue (TSA) complex containing a 3PG-AlF(4)(-)-ADP moiety. There are an overall increases in amide protection from hydrogen exchange when the protein binds the substrate and product ligands and an additional increase when the TSA complex is formed. This is consistent with stabilisation of the protein structure by ligand binding. However, there is no correlation between the chemical shift changes and the protection factor changes, indicating that the protection factor changes are not associated with an overall shortening of hydrogen bonds in the protected ground state, but rather can be ascribed to the properties of the high-energy, exchange-competent state. Therefore, an overall structural tightening mechanism is not supported by the data. Instead, we observed that some cooperativity is exhibited in the N-domain, such that within this domain the changes induced upon forming the TSA complex are an intensification of those induced by binding 3PG. Furthermore, chemical shift changes induced by 3PG binding extend through the interdomain region to the C-domain beta-sheet, highlighting a network of hydrogen bonds between the domains that suggests interdomain communication. Interdomain communication is also indicated by amide protection in one domain being significantly altered by binding of substrate to the other, even where no associated change in the structure of the substrate-free domain is indicated by chemical shifts. Hence, the communication between domains is also manifested in the accessibility of higher-energy, exchange-competent states. Overall, the data that are consistent with structural tightening relate to defined regions and are close to the 3PG binding site and in the hinge regions of 3-phosphoglycerate kinase.
机译：已经测量了酰胺磷酸酶化学位移和氢交换速率随磷酸甘油酸激酶在其催化循环中的变化，以评估它们是否与蛋白质中氢键的变化相关。比较了四个代表性状态：游离酶，包含3-磷酸甘油酸（3PG）的产物复合物，包含ADP的底物复合物和包含3PG-AlF（4）（-）-ADP的过渡态类似物（TSA）复合物部分。当蛋白质结合底物和产物配体时，酰胺保护免受氢交换的总体增加，而当形成TSA复合物时，另外的增加。这与通过配体结合来稳定蛋白质结构相一致。但是，化学位移的变化与保护因子的变化之间没有相关性，这表明保护因子的变化与保护基态下氢键的整体缩短无关，而可以归因于高保护基的性质。能量，具有交换能力的状态。因此，数据不支持总体的结构收紧机制。取而代之的是，我们观察到在N域中表现出一些合作性，因此在该域中，形成TSA复合物时诱导的变化是结合3PG诱导的变化的增强。此外，由3PG结合引起的化学位移变化通过域间区域扩展到C域β-折叠，从而突出表明域之间氢键的网络，表明域间通信。域间通讯也通过酰胺保护表示，一个域中的酰胺保护被底物与另一域的结合显着改变，即使在无底物域结构没有相关变化的情况下，化学位移也没有显示。因此，域之间的通信也表现为高能量，具有交换能力的状态的可访问性。总体而言，与结构收紧相符的数据涉及定义的区域，并且靠近3PG结合位点和3-磷酸甘油酸激酶的铰链区域。

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