Conformational linkage during catalysis by TrpRS

TrpRS 催化过程中的构象连接

• 批准号: 6796746
• 负责人: Charles W. Carter
• 金额: $ 26.15万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-15 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6796746
• 关键词: Bacillus stearothermophilus; SDS polyacrylamide gel electrophoresis; X ray crystallography; active sites; acylation; adenosine triphosphate; aminoacid tRNA ligase; aminoacyl tRNA; calorimetry; computer simulation; conformation; drug discovery /isolation; enzyme activity; enzyme mechanism; enzyme structure; molecular dynamics; protein structure function; thermodynamics; tryptophan

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.

描述（由申请人提供）：先前支持的工作提供了源自前所未有的证明，即氨基酸链球菌丁香烷基苯基苯基-TRNA合成型（TRPRS）激活氨基酸的过渡状态（TRPRS）与重要的域运动相吻合，可将磷酸盐结合的子体固定在不适合腺苷的子体中。此外，域运动还恢复了严重的侧链堆积关系，这些关系被破坏并破坏了过渡前的态度构象。因此，活性位点确实为过渡状态构型提供了静态模具，就像它在酶催化的普遍接受模型中一样。相反，蛋白质构象本身可能发挥前所未有的作用。因此，TRPR是研究如何将催化与蛋白质构象相关的绝佳模型。 结构反应曲线中最明显的差距包括过渡状态本身的结构以及与酰基转移和产品释放有关的tRNA复合物，现在可以解决晶体。一个关键的难题是过渡态下腺嘌呤核苷酸的结构，可以通过提出的动力学同位素测量来访问。最后，现在掌握的结构数据以及所提出的新结构建立了引人注目的类比，在TRPR催化过程中重要分子内相互作用与寡聚蛋白中的变构行为之间存在。这些证明了对热力学关系的详细研究是合理的，该研究将不同的配体与特定蛋白质构象转变及其在催化中的作用联系起来。我们进一步提议通过热力学测量（量热法）和理论研究（分子动力学模拟）评估这些关系，以提供广泛的，重叠的平衡常数和焓集，并从中可以从中从中开发出热力学链接关系的综合记录。 酶促循环至少涉及至少三个不同的TRPR构象 - 开放，封闭，过渡前和产品 - 与在肌球蛋白和F1 ATPase（例如肌球蛋白和F1 ATPase）中观察到的类似的三个构型同源。 TRPR中的地面不稳定可能具有与此类酶相似现象的相似现象的密切类比。