Storage and Recovery of ATP binding energy in Metal-Catalyzed Phosphoryl-Transfer

金属催化磷酰基转移中 ATP 结合能的储存和回收

• 批准号: 8195178
• 负责人: Charles W. Carter
• 金额: $ 4.33万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8195178
• 关键词: Acceleration; Accounting; Active Sites; Affect; Affinity; Amino Acids; Amino Acyl-tRNA Synthetases; Area; Bacillus stearothermophilus; Behavior; Binding; Binding Sites; Biological Assay; Biological Models; Biomechanics; Catalysis; Charge; Chemicals; Complex; Coupled; Coupling; Data; Data Set; Drug Industry; Effectiveness; Elements; Enzymatic Biochemistry; Enzymes; Equilibrium; Experimental Designs; Family; Free Energy; Genotype; Goals; Guanosine Triphosphate Phosphohydrolases; Hydrolysis; Ions; Kinetics; Ligands; Link; Location; Measures; Mediating; Metals; Methods; Modeling; Molecular; Molecular Conformation; Motion; Motor; Movement; Mutagenesis; Mutation; N-terminal; Nucleotides; Polymerase; Positioning Attribute; Property; Proteins; Purines; Recovery; Regulation; Relative (related person); Relaxation; Research; Role; Sampling; Signal Transduction; Signaling Protein; Simulate; Site; Specificity; Structure; Substrate Specificity; Testing; Thermodynamics; Training; Tryptophan; Tryptophan-tRNA Ligase; Tyrosine; Validation; Work; combinatorial; conformational alteration; conformational conversion; design; dimer; drug development; innovation; insight; mutant; novel; predictive modeling; programs; protein function; public health relevance; purine; research study; statistics; structural biology; tripolyphosphate; tryptophyltyrosine

DESCRIPTION (provided by applicant): Most drug development targets catalyze phosphoryl-transfer to or from nucleotide triphosphates. Because catalysis changes their conformation, both affinity and selectivity for these targets depend on structural aspects that are changing rapidly precisely as they develop highest affinity. Thus, they are, necessarily, "moving targets". Many such enzymes also transduce chemical free energy by linking hydrolysis of their purine triphosphate substrates to conformational changes used for cellular work and signaling. These enzymes include many that possess 1/2 folds described by Rossmann. Virtually all use a metal ion for catalysis. Our central hypothesis is that in enzymes whose conformational changes are responsible for free energy transduction the metal acts catalytically if, and only if, conformational changes reposition it. More formally, interactions of the Mg2+ ion from within the active site oppose catalysis, while longer-range interactions drive conformational motions from elsewhere in the protein, acting indirectly to change the Mg2+ coordination so that it can stabilize the chemical transition state. Preliminary work on B. stearothermophilus tryptophanyl-tRNA synthetase, TrpRS, shows conclusively that active-site protein-metal coupling opposes catalysis, in keeping with the hypothesis. To confirm the hypothesis, we seek positive evidence demonstrating synergistic interactions with the metal from a specific and highly conserved packing motif (the D1 Switch) common to all Rossmannoid enzymes (Aim 1). Thermodynamic cycles for several D1 point mutants, assayed with Mg2+ and Mn2+ have demonstrated significant synergistic coupling to the catalytic metal. A complete dataset may also support specific molecular mechanisms for this long-range coupling, thereby strengthening the hypothesis and broadening its impact on understanding molecular mechanisms of free- energy transduction. We discovered in preliminary work that Mn2+ also relaxes specificity of TrpRS for Trp vs. Tyr. In Aim 2, we will examine D1 (Aim 1) and D3 (specific to the Trp pocket) switch mutants to determine if this effect requires long-range coupling or arises only from properties of the metal. Insight into the mechanism of Mn2+-induced relaxation of specificity may have important implications for understanding the mutagenic affect of Mn2+ in polymerases. Finally, TrpRS also provides a superb model system to test whether or not incomplete factorial experimental design can reduce the total number of experiments necessary to parameterize predictive models for how allosteric protein functions change with combinatorial mutations (Aim 3). If we can draw valid, useful conclusions about the complex behavior of the D1 Switch from a small subset of the full factorial design of 127 genotypes, using similar innovative designs will enhance the experimental characterization of both related (a similar switch exists in CheY) and dissimilar phenomena. PUBLIC HEALTH RELEVANCE: A pervasive and unsolved problem in structural biology is how catalysis of purine triphosphate hydrolysis is coupled to conformational changes necessary for specificity, regulation, signaling, and biomechanics. Our work has raised a new possibility of an unexpected and potentially widespread coupling mechanism whereby Mg2+ can act catalytically if and only if the conformation changes. Testing this hypothesis by combinatorial mutagenesis a widely conserved conformational switching motif in Bacillus stearothermophilus Tryptophanyl-tRNA synthetase will likely establish new mechanistic paradigms linking transition-state stabilization by Mg2+ to domain movement via distributed use of ATP binding energy, with broad relevance to catalysis specificity, and free-energy transduction.

描述（由申请人提供）：大多数药物开发目标催化磷酰基转移至三磷酸核苷酸或从三磷酸核苷酸转移。由于催化作用改变了它们的构象，因此这些靶标的亲和力和选择性都取决于结构方面，而这些结构方面随着它们发展出最高亲和力而精确地快速变化。因此，它们必然是“移动目标”。许多此类酶还通过将其三磷酸嘌呤底物的水解与用于细胞工作和信号传导的构象变化联系​​起来来转导化学自由能。这些酶包括许多具有 Rossmann 描述的 1/2 折叠的酶。事实上，所有这些都使用金属离子进行催化。我们的中心假设是，在构象变化负责自由能转导的酶中，当且仅当构象变化重新定位时，金属才会发挥催化作用。更正式地说，活性位点内 Mg2+ 离子的相互作用对抗催化作用，而长程相互作用则驱动蛋白质其他位置的构象运动，间接改变 Mg2+ 配位，从而稳定化学过渡态。对嗜热脂肪芽胞杆菌色氨酸-tRNA 合成酶 TrpRS 的初步研究最终表明，活性位点蛋白质-金属偶联会阻碍催化作用，这与假设一致。为了证实这一假设，我们寻求积极的证据，证明所有 Rossmannoid 酶共有的特定且高度保守的包装基序（D1 开关）与金属之间存在协同相互作用（目标 1）。用 Mg2+ 和 Mn2+ 测定的几种 D1 点突变体的热力学循环已证明与催化金属具有显着的协同耦合作用。完整的数据集还可以支持这种长程耦合的特定分子机制，从而加强假设并扩大其对理解自由能转导分子机制的影响。我们在前期工作中发现，Mn2+ 还放宽了 TrpRS 对 Trp 与 Tyr 的特异性。在目标 2 中，我们将检查 D1（目标 1）和 D3（特定于色氨酸口袋）开关突变体，以确定这种效应是否需要长程耦合或仅由金属的特性产生。深入了解 Mn2+ 诱导的特异性松弛机制可能对于理解 Mn2+ 在聚合酶中的诱变作用具有重要意义。最后，TrpRS 还提供了一个出色的模型系统，用于测试不完整的因子实验设计是否可以减少参数化预测模型所需的实验总数，以了解变构蛋白功能如何随组合突变而变化（目标 3）。如果我们能够从 127 个基因型的完整析因设计的一小部分中得出有关 D1 开关复杂行为的有效、有用的结论，那么使用类似的创新设计将增强相关现象（CheY 中存在类似开关）和不同现象的实验表征。 公共健康相关性：结构生物学中一个普遍且尚未解决的问题是三磷酸嘌呤水解的催化如何与特异性、调节、信号传导和生物力学所需的构象变化耦合。我们的工作提出了一种意想不到的、潜在广泛的耦合机制的新可能性，即当且仅当构象发生变化时，Mg2+ 才能发挥催化作用。通过组合诱变测试这一假设，嗜热脂肪芽孢杆菌色氨酸-tRNA合成酶中广泛保守的构象转换基序可能会建立新的机制范例，通过分布式使用ATP结合能将Mg2+的过渡态稳定与结构域运动联系起来，与催化特异性和自由能转导具有广泛的相关性。