Crystallographic Studies Of Biological Intermediates

描述（由申请人提供）：该补助金支持所有研究 斯托达德实验室进行的结构酶学活动。三 该赠款涵盖的相互关联的项目包括：1.结构 新型生物催化剂的测定和功能研究，主要是 核酸内切酶和剪接因子编码在移动的内含子内。在 上一个资助期，I-CreI和I-PpoI的结构 测定游离的和与DNA结合的核酸内切酶。其他研究 描述了位点识别的催化机理和热力学， 还报道了I-PpoI的结合。斯托达德博士建议延长他的 在下一个拨款期内，就这两个系统进行研究，并决定 来自相关酶家族的几种新蛋白质的结构：I-AniI， I-NanI，I-SspI和I-Tevill。2.持续发展的时间分辨 晶体学方法来确定催化剂的结构 中间体，并研究基于这些结构的反应曲线。在 上一个资助期，使用光解触发，劳厄衍射， 使用几种模型系统报道了诱变和溶剂交换。 Stoddard博士建议使用三种模型系统继续这些研究： I-PpoI、锤头状核酶和鸟氨酸氨基转移酶。斯托达德博士 还提出了开发方法来模拟反应坐标和自由 单个催化反应步骤的能量分布，使用以下结构： 离散的中间体作为定义端点的定义明确的点， 通路 3.利用变构调节位点作为基于结构的靶点 药物设计在上一个资助期内， 丙酮酸激酶的变构调节，其次是 生物化学、遗传学和计算研究证明了 PK失调对细胞周期进程的影响以及鉴定出的新的合成 酶的变构效应物。斯托达德博士建议继续这些 通过对PK的人R和M2同工酶进行类似实验的研究， 和人核糖核苷酸还原酶。

DESCRIPTION (provided by applicant): This grant supports all research activities in structural enzymology carried out by the Stoddard lab. Three interconnected projects are covered under this grant: 1. Structure determination and functional studies of novel biological catalysts, primarily endonucleases and splicing factors encoded within mobile introns. In the previous funding period, the structure of the I-CreI and I-PpoI homing endonucleases were determined, both free and bound to DNA. Additional studies describing the catalytic mechanism and thermodynamics of site recognition and binding for I-PpoI were also reported. Dr. Stoddard proposes to extend his studies on these two systems in the next funding period, and to determine the structures of several new proteins from related enzyme families: I-AniI, I-NanI, I-SspI and I-Tevill. 2. Continued development of time-resolved crystallographic methods to determine the structures of catalytic intermediates, and to study reaction profiles based on these structures. In the previous funding period, studies using photolytic triggering, Laue diffraction, mutagenesis, and solvent exchange were reported using several model systems. Dr. Stoddard proposes to continue these studies using three model systems: I-PpoI, the hammerhead ribozyme and ornithine aminotransferase. Dr. Stoddard also proposes to develop methods to model the reaction coordinates and free energy profiles of individual catalytic reaction steps, using structures of discrete intermediates as well-defined points that define the ends of the pathway. 3. Exploitation of allosteric regulatory sites as targets for structure-based drug design. In the previous funding period, structural studies of the allosteric regulation of pyruvate kinase were reported, followed by biochemical, genetic and computational studies that demonstrate the effect of PK deregulation on cell cycle progression and that identified novel, synthetic allosteric effectors of the enzyme. Dr. Stoddard proposes to continue these studies by performing similar experiments on the human R and M2 isozymes of PK, and on human ribonucleotide reductase.