CAUSE AND EFFECT OF DIMER ASYMMETRY IN MERCURIC REDUCTAS

汞还原中二聚体不对称的原因和影响

• 批准号: 2188641
• 负责人: Susan Mary Miller
• 金额: $ 9.01万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-01-01 至 1999-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2188641
• 关键词: Bacillus; X ray crystallography; bacterial proteins; chemical binding; chemical kinetics; dimer; enzyme mechanism; enzyme model; enzyme structure; ligands; mercury; molecular site; mutant; oxidoreductase; site directed mutagenesis

The proposed research lies in the area of mechanistic enzymology, with long term goals of elucidating how protein structural elements or motifs specifically contribute to catalytic efficiency. Of particular interest is the study of molecular mechanisms and functional consequences of communication between remote ligand binding sites in proteins. For a wide variety of receptors, enzymes and binding proteins, signalling between distant sites is critical to the physiological function. Consequently, disruptions of site-site communication result in defective protein function, which almost certainly cause disease states if the defect is severe. Thus, identifying structural motifs and/or molecular networks for different types of intersite communication could provide valuable insight into disease-causing mutations, as well as suggest alternative strategies for designing drugs to control protein function. From a mechanistic view, the most poorly understood type of intersite communication is negative cooperativity between identical sites on homooligomers. In an alternating sites hypothesis, originally proposed for ATP synthases and, more recently for mercuric reductase, the functional role ascribed to negative cooperativity is one of switching the affinity for ligands at identical sites of a homooligomer in a concerted fashion to facilitate different steps of a catalytic pathway. Substantial evidence supports this hypothesis in the ATP synthases, but structural studies are difficult in that system. By contrast, mercuric reductases are soluble homodimers; genes for enzyme from two sources are overexpressed; and the crystal structure of one of those has been solved, all of which make this enzyme an attractive model system to explore the mechanistic significance and molecular basis of homooligomer asymmetry resulting from negative cooperative communication. Specifically, the predictions of the alternating sites hypothesis, as described for mercuric reductase will be tested using a combination of equilibrium titrations and single turnover kinetics on a set of mutants lacking one or more ligands in the Hg(II) binding sites. Additionally, spectrally characterized Hg(II) complexes of wild type and mutant enzymes will be structurally characterized using XAFS methods. Crystal structure data will be used to identify possible pathways for communication, which will then be explored through site- directed mutagenesis and complete physical and mechanistic characterization of the novel mutant enzymes.

拟议的研究在于机械酶学领域， 阐明蛋白质结构元素或基序的长期目标 特别有助于催化效率。 特别感兴趣 是分子机制的研究和功能后果 蛋白质中远程配体结合位点之间的通信。 宽阔 各种受体，酶和结合蛋白，信号传导 远处对生理功能至关重要。 最后， 现场通信的破坏导致蛋白质有缺陷 功能，几乎可以肯定会导致疾病状态是否缺陷 严重。 因此，确定结构基序和/或分子网络 不同类型的场所交流可以提供宝贵的见解 进入引起疾病的突变，并提出替代策略 用于设计药物以控制蛋白质功能。 从机械角度来看 最了解的类型的场所交流是负面的 家族植物的相同站点之间的合作性。 交替 地点假设，最初是为ATP合酶提出的，最近提出的 对于汞还原酶，功能作用归因于负 合作性是切换配体的亲和力之一 以一种协同的方式来促进家园的遗址 催化途径的步骤。 大量证据支持这一点 ATP合酶中的假设，但结构研究很难 该系统。 相比之下，汞还原酶是可溶的同二聚体。 来自两个来源的酶的基因过表达。和水晶 其中一个的结构已解决，所有这些都使该酶 一个有吸引力的模型系统，可探索机械意义和 负面的家族不对称性的分子基础 合作交流。 具体而言，对 如汞还原酶所述的交替位点假设将是 使用平衡滴定和单个周转率的组合进行了测试 在HG中缺少一个或多个配体的一组突变体上的动力学（II） 绑定位点。 另外，频谱表征的Hg（II）复合物的复合物 野生型和突变酶将在结构上使用XAF进行结构表征 方法。 晶体结构数据将用于识别可能 通信途径，然后将通过网站探索 - 定向诱变和完整的物理和机械 新型突变酶的表征。