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

描述(申请人提供)：大多数药物开发目标催化磷酸转移到或从核苷酸三磷酸盐。由于催化作用会改变它们的构象，因此这些靶标的亲和力和选择性都取决于结构方面，这些方面随着它们发展出最高的亲和力而迅速变化。因此，他们必然是“移动的目标”。许多这样的酶还通过将它们的三磷酸嘌呤底物的水解与用于细胞工作和信号传递的构象变化联系起来来转换化学自由能。这些酶包括许多具有罗斯曼所描述的1/2倍的酶。几乎所有的催化剂都使用金属离子进行催化。我们的中心假设是，在其构象变化负责自由能传递的酶中，当且仅当构象变化重新定位金属时，金属才起催化作用。更正式地说，活性中心内的镁离子相互作用反对催化，而较长距离的相互作用则驱动蛋白质其他地方的构象运动，间接地改变镁离子的配位，使其能够稳定化学过渡态。对嗜热脂肪芽孢杆菌色氨酸tRNA合成酶TrpRS的初步研究表明，活性部位的蛋白-金属偶联与催化相反，这与假设一致。为了证实这一假设，我们寻找了积极的证据，证明了与金属的协同作用来自一个特定和高度保守的包装基序(D1开关)，所有Rossmannoid酶(目标1)都共有。用Mg2+和Mn2+对几个D1点突变体的热力学循环进行了分析，结果表明它们与催化金属有显著的协同偶联作用。一个完整的数据集也可能支持这种远程耦合的特定分子机制，从而加强了这一假说，并扩大了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+还可以松弛TrpRs对Trp和Tyr的特异性。在目标2中，我们将研究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远程耦合，还是只由金属的性质引起。深入了解Mn2+诱导的特异性松弛机制对于理解Mn2+对聚合酶的诱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对变构蛋白质功能如何随组合突变而变化的预测模型进行参数化所需的实验总数(目标3)。如果我们能够从127个基因型的全析因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，使用类似的创新设计将增强对相关(CHEY中存在类似开关)和不同现象的实验表征。 公共卫生相关性：结构生物学中一个普遍存在且尚未解决的问题是，三磷酸嘌呤水解酶的催化如何与特异性、调节、信号和生物力学所需的构象变化相结合。我们的工作提出了一种新的可能性，即一种意想不到的、可能广泛存在的耦合机制，在这种机制下，当且仅当构象改变时，镁离子才能起催化作用。通过组合突变验证这一假说，脂肪嗜热芽孢杆菌色氨酸tRNA合成酶中一个广泛保守的构象转换基序可能会建立新的机制范例，将镁离子稳定的过渡态与结构域的移动联系起来，通过分布使用ATP结合能，与催化专一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.