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

描述（由申请人提供）：大多数药物开发靶点催化磷酸转移至核苷酸三磷酸或从核苷酸三磷酸转移。由于催化作用改变了它们的构象，因此对这些靶点的亲和力和选择性都取决于结构方面，这些结构方面在它们发展最高亲和力时精确地迅速变化。因此，它们必然是“移动目标”。许多这样的酶也通过将它们的嘌呤三磷酸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降低化学自由能。这些酶包括许多具有1/2折叠的酶，其描述如下： 罗斯曼几乎所有的催化剂都使用金属离子。我们的中心假设是，在其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金属时，金属起催化作用。更正式地说，来自活性位点内的Mg 2+离子的相互作用对抗催化作用，而长程相互作用驱动来自蛋白质其他部位的构象运动，间接作用改变Mg 2+配位，使其稳定化学过渡态。B的准备工作。嗜热脂肪菌双氢酰-tRNA合成酶TrpRS确凿地表明活性位点蛋白质-金属偶联对抗催化作用，与该假说一致。为了证实这一假设，我们寻求积极的证据，证明协同相互作用的金属从一个特定的和高度保守的包装基序（D1开关）共同的所有Rossmannoid酶（目的1）。用Mg 2+和Mn 2+测定的几个D1点突变体的热力学循环已经证明了与催化金属的显著协同偶联。完整的数据集还可以支持这种长程耦合的特定分子机制，从而加强该假设并扩大其对理解自由能转导的分子机制的影响。我们在初步工作中发现，Mn 2+也松弛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Trp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性质引起。深入了解Mn 2+诱导的特异性松弛的机制可能对理解Mn 2+在聚合酶中的致突变作用具有重要意义。最后，TrpRS还提供了一个极好的模型系统来测试不完全析因实验设计是否可以减少参数化预测模型所需的实验总数，以确定变构蛋白功能如何随着组合突变而变化（目标3）。如果我们能从127个基因型的全析因设计的一个小子集中得出关于D1开关复杂行为的有效、有用的结论，使用类似的创新设计将增强相关（CheY中存在类似开关）和不相似现象的实验表征。 公共卫生相关性：结构生物学中一个普遍而未解决的问题是嘌呤三磷酸水解的催化如何与特异性、调节、信号传导和生物力学所必需的构象变化相耦合。我们的工作提出了一种新的可能性，一个意想不到的和潜在的广泛的耦合机制，即Mg 2+可以发挥催化作用，当且仅当构象变化。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将可能建立新的机制范式，将Mg 2+通过分布使用ATP结合能将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。

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.