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

描述（由申请人提供）：大多数药物开发目标催化磷酸转移到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，这些靶标的亲和力和选择性都取决于它们的结构方面，而这些结构方面正是在它们发展出最高亲和力时迅速变化的。因此，它们必然是“移动的目标”。许多这类酶还通过将其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传递化学自由能。这些酶包括许多具有Rossmann描述的1/2折叠的酶。几乎所有的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，金属当且仅当构象变化使其重新定位时起催化作用。更正式地说，活性位点内的Mg2+离子相互作用反对催化作用，而较长距离的相互作用驱动蛋白质其他部位的构象运动，间接地改变Mg2+配位，从而稳定化学过渡态。初步工作b stearothermophilus tryptophanyl-tRNA合成酶、TrpRS明确地显示活性部位protein-metal耦合反对催化,与假设一致。为了证实这一假设，我们从所有Rossmannoid酶共有的一个特定且高度保守的包装基序（D1 Switch）中寻找积极的证据来证明与金属的协同作用（Aim 1）。用Mg2+和Mn2+测定了几个D1点突变体的热力学循环，结果表明它们与催化金属具有显著的协同耦合作用。完整的数据集还可以支持这种远程耦合的特定分子机制，从而加强假设并扩大其对理解自由能转导的分子机制的影响。我们在前期工作中发现，Mn2+也能放松trpr对Trp和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远程耦合或仅由金属的性质引起。深入了解Mn2+诱导特异性松弛的机制可能对理解Mn2+在聚合酶中的致突变作用具有重要意义。最后，trpr还提供了一个极好的模型系统，用于测试不完全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，以预测变构蛋白功能如何随组合突变而变化（目的3）。如果我们能够从127个基因型全因子设计的一小部分中得出关于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（CheY中存在类似开关）和不相似现象的实验表征。

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.