CAUSE AND EFFECT OF DIMER ASYMMETRY IN MERCURIC REDUCTAS

拟议的研究属于机械酶学领域，具有 阐明蛋白质结构元素或基序的长期目标 特别是对催化效率有贡献。特别感兴趣的 是研究血管紧张素转换酶的分子机制和功能后果 蛋白质中远程配体结合位点之间的通讯。对于一个宽广的 多种受体、酶和结合蛋白，在 距离较远的部位对生理功能至关重要。因此， 位点间通讯中断导致蛋白质缺陷 功能，这几乎肯定会导致疾病状态，如果缺陷是 很严重。因此，识别结构基序和/或分子网络 不同类型的站点间通信可以提供有价值的见解 致病突变，以及提出替代策略 用于设计控制蛋白质功能的药物。从机械论的观点来看， 人们最不了解的站点间通信类型是负面的 同源低聚物上相同位点之间的协同作用。以交替的方式 位点假说，最初是针对ATP合成酶提出的，最近又提出了 对于汞还原酶，其功能作用归因于负性 协作性是将配体的亲和力转换为相同的亲和力 同源齐聚物的位置以一致的方式促进不同的 催化途径的步骤。大量证据支持这一点。 假说，但结构研究是困难的 那个系统。相比之下，汞还原酶是可溶的同源二聚体； 来自两个来源的酶基因过度表达；晶体 其中一种的结构已经被解决，所有这些都使这种酶 一个吸引人的模型系统，以探索机械意义和 负性导致齐聚物不对称性的分子基础 合作沟通。具体地说， 交替位置假说，如对汞还原酶的描述，将是 使用平衡滴定和单次周转相结合的方法进行测试 汞(II)中缺少一个或多个配体的突变体的动力学 结合部位。此外，光谱表征的汞(II)配合物 野生型和突变型酶将使用XAFS进行结构表征 方法：研究方法。晶体结构数据将被用来确定可能的 交流的途径，然后将通过网站探索- 定向诱变与完全物理和机械化 新型突变酶的特性研究。

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.