RICIN--MECHANISM, TRANSITION STATE AND INHIBITOR DESIGN

蓖麻毒素是一种在蓖麻豆中发现的RNA净化毒素。单人间 腺嘌呤从28S rRNA中水解物，使核糖体 处于非活动状态。一种分子对哺乳动物细胞是致命的，使其成为 毒性最强的生物分子。蓖麻毒素的细胞毒性现在是 在临床试验中被利用来摧毁不需要的细胞。尽管 蓖麻毒素的新催化特性和潜在的临床应用 已知其底物专一性、催化机理或转化 国家结构。Km/Ki大于1的唯一缓蚀剂 都是这个实验室准备的。 动力学同位素效应在核磁共振研究中的应用进展 酶反应使主要的 几种N-核糖水解酶过渡态的特性。蓖麻毒素 A链催化的反应在化学上类似于核苷和 核苷酸N-核糖水解酶，是类似分析的候选者。 过渡状态信息的可用性证明是基本的 关于催化机理的信息，并在化学上提供了帮助 三聚氰胺催化亚基的机理和过渡态结构 蓖麻毒素，蓖麻毒素A链。这些信息将被应用于 这些分子是酶的抑制物。建议使用的抑制剂 将被合成并表征为动力学和结合 实验。茎环RNA和杂交抑制剂分子将是 合成以确定底物和抑制剂的特异性。选定的阀杆- 环RNA结构将通过核磁共振来解决。加入了一个自旋- 将在茎环RNA类似物的去嘌呤位置使用标记 以提供结合探针并允许绘制蛋白质-RNA图谱 催化部位空腔中的几何形状。过渡状态结构将 通过动力学同位素效应进行研究。底物RNA类似物 将被合成以测试N-核糖键的溶剂分解是否依赖于 关于离开基团的活化(酸催化的溶剂分解)、核糖活化 (核氧卡宾离子稳定)或核糖羟基电离 (碱催化溶解)或以上一种的组合 机械装置。紧密结合的抑制剂的特征是 与蓖麻毒素A链共结晶用于X射线晶体研究。

Ricin is an RNA-depurinating toxin found in the castor bean. A single adenine is hydrolyzed from 28S rRNA, rendering the ribosome inactive. One molecule is lethal for mammalian cells, making it one of the most toxic biological molecules. The cytotoxicity of ricin is now being exploited in clinical trials to destroy unwanted cells. Despite the novel catalytic properties and potential clinical uses of ricin, little is known of it's substrate specificity, catalytic mechanism or transition state structure. The only inhibitors with Km/Ki greater than 1 have been prepared by this laboratory. Recent advances in the application of kinetic isotope effects to enzymatic reactions has permitted the characterization of the major features of several N-ribohydrolase enzymatic transition states. Ricin A-chain catalyzes a reaction chemically similar to nucleoside and nucleotide N-ribohydrolases and is a candidate for similar analysis. Availability of transition state information proves fundamental information of the catalytic mechanism and has assisted in chemical mechanisms and transition state structure for the catalytic subunit of ricin, ricin A chain. This information will be applied to the design of molecules which are inhibitors of the enzyme. The proposed inhibitors will be synthesized and characterized by kinetic and binding experiments. Stem-loop RNA and hybrid inhibitor molecules will be synthesized to define substrate and inhibitor specificity. Selected stem- loop RNA structures will be solved by NMR. Incorporation of a spin- label at the depurination site of stem-loop RNA analogues will be used to provide a binding probe and to permit mapping of the protein-RNA geometry in the catalytic site cavity. The transition state structure will be investigated by kinetic isotope effects. Substrate RNA analogues will be synthesized to test if solvolysis of the N-ribosidic bond depends on leaving group activation (acid-catalyzed solvolysis), ribosyl activation (ribooxocarbenium ion stabilization) or ribosyl hydroxyl ionization (base-catalyzed solvolysis) or a combination of more than one of these mechanisms. Inhibitors which bind tightly will be characterized by cocrystalization with ricin A-chain for x-ray crystal studies.