STRUCTURE AND REACTIVITY OF METALLOTHIONEIN

这项提案描述了一系列旨在阐明 金属硫蛋白中多金属离子的配位环境 并表征了它们最近发现的化学反应能力。自.以来 这种富含巯基的金属结合蛋白是一种正常的细胞成分 在人类和几乎所有其他有机体中，都可以由 重金属，包括镉的管理，以及各种 生理应激中，金属硫蛋白被认为起着重要的作用 在锌和铜代谢、重金属解毒和 有机体应激反应。各种物理化学技术将 被用来阐明详细的结构-功能关系。我们的 主要的结构工具将是~(113)Cd核磁共振，这是一个独特的强大的 金属硫蛋白结构的探索及其最近用于证明 镉被分成两个离散的金属硫酸盐簇合物 蛋白。两群集安排的一般性将通过以下方式进行测试 几种金属硫蛋白的~(113)Cd核磁共振参数研究 来源多样。结合~(13)C核磁共振研究，配位球 镉和锌的特性将在各种不同的 条件。详细的结构、动力学和热力学研究 金属离子和载脂蛋白形成金属硫蛋白的过程， 硫蛋白，将为其在体内的可能机制提供洞察力 集合。金属离子交换的动力学和机理研究 硫蛋白结合锌和镉，并进行自由离子， 平行~(113)Cd核磁共振法研究化合物的差示反应活性 团簇到金属交换。金属硫蛋白的新反应 包括锌向脱碳酸酐酶的转移，EDTA提取 金属离子，硫醇酸盐与DTNB的二硫键交换反应 将重新调查以确定金属团簇之间的关系 对反应动力学的影响。新型不对称二硫醇镉配合物 将合成螯合配体作为~(113)核磁共振研究的模型化合物。

This proposal describes a series of investigations designed to elucidate the coordination environments of the multiple metal ions in metallothionein and characterize their recently recognized chemical reactivities. Since this sulfhydryl-rich metalbinding protein is a normal cellular constituent in humans and virtually all other organisms and can be induced by the administration of heavy metals, including cadmium, and by a variety of physiological stresses, metallothionein is believed to play an important role in zinc and copper metabolism, heavy metal detoxification, and organismic stress response. A variety of physicochemical techniques will be utilized to elucidate detailed structure-function relationships. Our principal structural tool will be 113Cd NMR, which is a uniquely powerful probe of metallothionein structure and which was recently used to prove that cadmium is grouped in two discrete metalthiolate clusters in the protein. The generality of the two-cluster arrangement will be tested by investigating the 113Cd NMR parameters of metallothioneins from several diverse sources. In concert with 13C NMR studies, the coordination spheres of both cadmium and zinc will be characterized under a variety of conditions. Detailed structural, kinetic and thermodynamic investigations of the formation of metallothionein from metal ions and the apo-protein, thionein, will provide insights into the possible mechanism of its in vivo assembly. Kinetic and mechanistic studies of metal exchange between thionein-bound zinc and cadmium, and the free ions will be carried out, with parallel 113Cd NMR investigation of the differential reactivities of the clusters to metal exchange. Novel reactions of metallothionein including zinc transfer to apo-carbonic anhydrase, EDTA extraction of the metal ions, and disulfide interchange reaction of the thiolates with DTNB will be reinvestigated to determine the relationship of the metal clusters on the reaction kinetics. Novel cadmium complexes with assymetric dithiol chelating ligands will be synthesized as model compounds for 113NMR studies.