Conformational linkage during catalysis by TrpRS

描述（由申请人提供）：先前支持的工作提供了迄今为止前所未有的证明，即由B激活氨基酸的过渡态。嗜热脂肪芽孢杆菌β-氨基-tRNA合成酶（TrpRS）与一个显着的结构域运动，重新定位焦磷酸结合亚位点相对于腺苷亚位点，而不改变β-氨基-tRNA结合亚位点相一致。此外，域运动恢复显着的侧链包装的关系，这是中断和不稳定的前过渡态TrpRS构象。因此，活性位点确实为过渡态构型提供了一个静态模型，就像它在酶催化的普遍接受的模型中所做的那样。相反，蛋白质构象本身可能发挥前所未有的，更积极的作用。因此，TrpRS是研究催化作用如何与蛋白质构象联系起来的一个很好的模型。 结构反应谱中最明显的缺口包括过渡态本身的结构和涉及酰基转移和产物释放的tRNA复合物，现在可以解决晶体。一个关键的难题是腺嘌呤核苷酸在过渡态的结构，这是通过拟议的动力学同位素测量访问。最后，现在手头的结构数据，连同提出的新结构建立令人信服的类比存在重要的分子内相互作用在TrpRS催化和变构行为的寡聚蛋白。这些证明了一个详细的调查的热力学关系，连接不同的配体的结合特定的蛋白质构象转变，以及它们如何在催化作用。我们建议通过热力学测量（量热法）和理论研究（分子动力学模拟）进一步评估这些关系，以提供一个广泛的，重叠的平衡常数和平衡系数集，从中可以开发出一个综合的热力学联系关系。 酶促循环涉及至少三种不同的TrpRS构象-开放、封闭、过渡前状态和产物-这与在能量转导ATP酶如肌球蛋白和F1 ATP酶中观察到的三种构象的类似集合同源。TrpRS中的基态不稳定可能与此类酶中的类似现象具有密切的相似性。

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.