Crystallographic Studies Of Biological Intermediates

描述(由申请者提供)：这项资助支持所有研究 由斯托达德实验室进行的结构酶学活动。三 这笔赠款涵盖相互关联的项目：1.结构 新型生物催化剂的测定及功能初步研究 可移动内含子中编码的内切酶和剪接因子。在 上一个供资期间，综合研究和开发倡议的结构和综合政策倡议的归位 测定了游离型和结合型核酸内切酶。其他研究 描述了位识别和位识别的催化机理和热力学 还报道了与I-PpoI的结合。斯托达德博士提议延长他的 在下一个资助期就这两个系统进行研究，并决定 几种新的相关酶家族蛋白的结构：I-anii， I-Nani，I-Sspi和I-Tevill2.时间分辨的持续发展 确定催化剂结构的结晶学方法 中间体，并根据这些结构研究反应概况。在 以前的资助期，研究使用光解触发，劳厄衍射， 用几个模型系统报道了诱变和溶剂交换。 斯托达德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转氨酶。斯托达德博士 还建议开发方法来模拟反应坐标和自由 使用以下结构的各个催化反应步骤的能量分布 离散的中间体，作为定义 路径。 3.开发变构调控位点作为基于结构的靶点 药物设计。在上一个资助期内， 报道了丙酮酸激酶的变构调节，其次是 生物化学、遗传学和计算机研究证明了 PK对细胞周期进程的解除调控及其发现的新的、合成的 酶的变构效应。斯托达德博士建议继续这样做 通过对人类PK的R和M2同工酶进行类似的实验进行研究， 以及人类核糖核酸还原酶。

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.