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: 7993221
• 负责人: Charles W. Carter
• 金额: $ 38.38万
• 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/7993221
• 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）：大多数药物开发靶向催化磷酸化转移到核苷酸三磷酸盐。由于催化改变了它们的构象，因此对这些靶标的亲和力和选择性都取决于结构方面，这些方面随着它们发展最高亲和力而迅速变化。因此，它们必然是“移动目标”。许多这样的酶还通过将其嘌呤三磷酸盐底物的水解与用于细胞工作和信号传导的构象变化来传递化学自由能。这些酶包括罗斯曼描述的1/2倍的许多酶。几乎所有人都使用金属离子进行催化。我们的中心假设是，在酶变化的酶中，自由能转导的原因是金属在催化作用时，并且只有当构象变化重新定位时。更正式的是，MG2+离子从活性部位内部反对催化，而长距离相互作用驱动蛋白质中其他地方的构象运动，间接起作用以改变MG2+配位，从而可以稳定化学过渡态。 TRPRS的螺旋烷基 - tRNA合成酶的初步工作最终表明，活跃的位点蛋白质 - 金属偶联与假设相对于催化。为了证实该假设，我们寻求积极的证据，证明来自所有Rossmannoid酶共有的特定且高度保守的填充基序（D1开关）与金属的协同相互作用（AIM 1）。用Mg2+和MN2+测定的几个D1点突变体的热力学循环表现出与催化金属的显着协同耦合。完整的数据集还可以支持这种远程耦合的特定分子机制，从而增强了假设并扩大了其对理解自由能传输的分子机制的影响。我们在初步工作中发现了MN2+还放宽了TRP对TRP与Tyr的特异性。在AIM 2中，我们将检查D1（AIM 1）和D3（特定于TRP口袋）开关突变体，以确定此效果是否需要远距离耦合或仅来自金属的性质。洞悉MN2+诱导的特异性松弛机制可能对理解聚合酶中MN2+的诱变影响具有重要意义。最后，TRPR还提供了一个出色的模型系统来测试不完整的阶乘实验设计是否可以减少为变构蛋白功能如何随组合突变而变化所需的预测模型所需的实验总数（AIM 3）。如果我们可以得出关于从127个基因型的完整阶乘设计的一小部分转换D1转换的有效的，有用的结论，则使用相似的创新设计将增强相关的实验表征（Chey中存在相似的开关）和不同的现象。 公共卫生相关性：结构生物学中的普遍和未解决的问题是如何将三磷酸嘌呤水解的催化与特异性，调节，信号和生物力学所必需的构象变化结合在一起。我们的工作提出了一种新的可能性，具有出乎意料的广泛耦合机制，即MG2+在构象变化时才可以催化作用。通过组合诱变检验这一假设，一种在芽孢杆菌stearothermophilus throptypophanyl-tRNA合成酸酯中广泛保守的构象切换基序可能会建立通过MG2+与域通过域与ATP结合能量的分布式使用与cat catalsyss的分布式使用，从而建立新的机械范式，将MG2+与域运动通过域进行过渡稳定，并均匀相关性。