Conformational linkage during catalysis by TrpRS

描述(由申请人提供)：以前支持的工作提供了迄今为止前所未有的证明，即脂肪嗜热杆菌色氨酰-tRNA合成酶(TrpRS)激活氨基酸的过渡状态与显著的结构域运动一致，该运动相对于腺苷亚基重新定位焦磷酸结合亚基，而不改变色氨酸结合亚基。此外，结构域运动恢复了显著的侧链堆积关系，这些关系在过渡前状态TrpRS构象中被破坏和不稳定。因此，活性中心确实为过渡态构型提供了一个静态模型，就像在通常接受的酶催化模型中所做的那样。相反，蛋白质构象本身可能发挥着前所未有的、更活跃的作用。因此，TrpRS是研究催化作用如何与蛋白质构象相联系的一个很好的模型。 结构反应谱中最明显的差距包括过渡态本身的结构和参与酰基转移和产物释放的tRNA络合物的结构，这些结构的晶体现在都可以解决。一个关键的谜题是处于过渡态的腺嘌呤核苷酸的结构，这可以通过拟议的动力学同位素测量来获得。最后，现在的结构数据，加上建议的新结构，建立了令人信服的相似之处，在TrpRS催化过程中的重要分子内相互作用和寡聚体蛋白质的变构行为之间存在着相似之处。这些证明了对将不同配体的结合与特定蛋白质构象转变联系起来的热力学关系的详细研究，以及它们如何在催化中发挥作用。我们建议进一步通过热力学测量(量热法)和理论研究(分子动力学模拟)来评估这些关系，以提供一组广泛的、重叠的平衡常数和热焓，从中可以开发出热力学连接关系的完整帐户。 酶的循环至少包括三种不同的TrpRS构象-开放的、关闭的、过渡前状态和产物-它们与在能量转导ATPase中观察到的类似的三种构象同源，如肌球蛋白和F1 ATPase。TrpRs中的基态不稳定可能与此类酶中的类似现象有密切的相似之处。

DESCRIPTION (provided by applicant): Previously supported work provides a heretofore unprecedented demonstration that the transition state for amino acid activation by B. stearothermophilus tryptophanyl-tRNA synthetase (TrpRS) coincides with a significant domain motion that relocates the pyrophosphate binding subsite relative to the adenosine subsite, without changing the tryptophan-binding subsite. Moreover, the domain motion restores significant side-chain packing relationships which are disrupted in and destabilize the pre-transition state TrpRS conformation. Thus the active site does provide a static mould for the transition-state configuration as it does in commonly accepted models of enzymic catalysis. Rather, protein conformation may itself play an unprecedented, and more active role. TrpRS is thus an excellent model for investigating how catalysis can be linked to protein conformation. The most obvious gaps in the structure reaction profile include the structures of the transition state itself and tRNA complexes involved in acyl-transfer and product release, for which crystals are now available to be solved. A key puzzle is the structure of adenine nucleotide in the transition state, which is accessible via proposed kinetic isotope measurements. Finally, the structural data now in hand, together with the proposed new structures establish compelling analogies exist between important intramolecular interactions during TrpRS catalysis and allosteric behavior in oligomeric proteins. These justify a detailed investigation of the thermodynamic relationships that link the binding of different ligands to specific protein conformational transitions, and how they function in catalysis. We propose further to assess these relationships by thermodynamic measurements (calorimetry) and theoretical studies (molecular dynamics simulations) to provide an extensive, overlapping set of equilibrium constants and enthalpies from which an integrated account of thermodynamic linkage relationships can be developed. The enzymatic cycle involves at least three distinct TrpRS conformations - open, closed, pre-transition state, and products - which are homologous to similar sets of three conformations observed in energy transducing ATPases like myosin and F1 ATPase. Ground-state destabilization in TrpRS likely has close analogies with similar phenomena in such enzymes.