Conformational linkage during catalysis by TrpRS

TrpRS 催化过程中的构象连接

• 批准号: 6945161
• 负责人: Charles W. Carter
• 金额: $ 24.84万
• 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/6945161
• 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迄今为止前所未有的证明，即嗜热脂肪芽孢杆菌色氨酰-tRNA合成酶（TrpRS）的氨基酸激活过渡态与显着的结构域运动一致，该结构域运动相对于腺苷重新定位焦磷酸结合亚位点亚位点，而不改变色氨酸结合亚位点。此外，域运动恢复了重要的侧链堆积关系，这些关系在过渡前状态 TrpRS 构象中被破坏并不稳定。因此，活性位点确实为过渡态构型提供了静态模型，就像在普遍接受的酶催化模型中一样。相反，蛋白质构象本身可能发挥着前所未有的、更积极的作用。因此，TrpRS 是研究催化如何与蛋白质构象联系起来的优秀模型。 结构反应曲线中最明显的差距包括过渡态本身的结构以及参与酰基转移和产物释放的 tRNA 复合物的结构，现在可以通过晶体来解决这些问题。一个关键的难题是过渡态腺嘌呤核苷酸的结构，可以通过所提出的动力学同位素测量来了解这一结构。最后，现有的结构数据以及提出的新结构在 TrpRS 催化过程中重要的分子内相互作用与寡聚蛋白的变构行为之间建立了令人信服的类比。这些证明了对不同配体的结合与特定蛋白质构象转变之间的热力学关系以及它们如何在催化中发挥作用的详细研究是合理的。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，以提供一组广泛的、重叠的平衡常数和焓，从中可以开发热力学联系关系的综合解释。 酶循环涉及至少三种不同的 TrpRS 构象——开放、闭合、过渡前状态和产物——它们与在能量转导 ATP 酶（如肌球蛋白和 F1 ATP 酶）中观察到的相似的三种构象组同源。 TrpRS 中的基态不稳定可能与此类酶中的类似现象非常相似。

项目成果

Did tRNA synthetase classes arise on opposite strands of the same gene?

• DOI: 10.1016/s1097-2765(02)00688-3
• 发表时间: 2002-10-01
• 期刊: MOLECULAR CELL
• 影响因子: 16
• 作者: Carter, CW;Duax, WL
• 通讯作者: Duax, WL

Coding of Class I and II Aminoacyl-tRNA Synthetases.

• DOI: 10.1007/5584_2017_93
• 发表时间: 2017
• 期刊: Advances in experimental medicine and biology
• 作者: Carter CW Jr

Structure alignment via Delaunay tetrahedralization.

通过 Delaunay 四面体化进行结构对齐。

• DOI: 10.1002/prot.20479
• 发表时间: 2005
• 期刊: Proteins
• 影响因子: 2.9
• 作者: Roach,Jeffrey;Sharma,Shantanu;Kapustina,Maryna;CarterJr,CharlesW
• 通讯作者: CarterJr,CharlesW