Conformational Coupling and the Basis for Metal Ion Specificity in Superoxide Dismutase

超氧化物歧化酶的构象偶联和金属离子特异性的基础

• 批准号: 9418181
• 负责人: Anne-Frances Miller
• 金额: $ 30.5万
• 依托单位: Johns Hopkins University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-01-01 至 1997-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9418181&HistoricalAwards=false
• 关键词: Conformational; Coupling; Basis; Metal; Ion

9418181 Miller The research will employ Fe and Mn-containing superoxide dismutase (SOD) as a model system and exploit the unique ability of nuclear magnetic resonance spectroscopy (NMR) to directly observe protein groups and monitor structure in solution. The objectives are to elucidate the conformational interplay between the metal ion site and the protein of SOD upon substrate analog binding to the metal ion, and to determine why FeSOD protein is inactive with Mn bound instead of Fe, and MnSOD protein is inactive with Fe bound instead of Mn. Isotope edited NMR will be used in conjunction with amino acid-specific isotopic labeling to produce drastically simplified spectra showing signals of labeled amino acids only. Comparisons of spectra of SOD with and without the substrate analog azide bound will identify amino acids affected by azide binding and indicate by what mechanisms the protein is conformationally responsive to events at the metal ion. By monitoring amino acids at the interfaces between domains or subunits, it will be possible to determine whether the conformation change involves potentially general mechanisms for conformational change including (1) relative motions of domains bridged by the metal ion, (2) relative motions of subunits and (3) propagation of conformational effects via hydrogen bonding networks. These studies build on the known structure for SOD and exploit the unique ability of NMR to directly observe protons, provide information on their participation in hydrogen bonds and provide qualitative and quantitative information on both structure and dynamics of proteins in solution unconstrained by crystal packing. The work will also determine why SODs containing the wrong metal ion are inactive, by evaluating their competence for individual elements of catalysis including each of the half reactions, the reduction midpoint potential, proton donation, substrate analog binding and active site structure, thus specifying the chemical reaso n for catalytic incompetence. Site-directed and random mutagenesis will be used to convert MnSOD protein into a FeSOD. Characterization of these SODs will reveal what amino acid changes are important, the extent to which these amino acid changes support activity with the wrong metal, and at what cost to activity with the correct metal. %%% Metalloenzymes combine the reactivity of metal centers with the exquisite specificity and control of enzymes. The Fe-containing superoxide dismutases (FeSODs) and the homologous Mn-containing superoxide dismutases (MnSODs) are metalloenzymes that catalyze the conversion of superoxide to dioxygen and hydrogen peroxide. SOD is relatively small, very stable and soluble but also embodies two central features of metalloenzyme catalysis: the metal site and the protein are believed to affect each other's structure, and each type of SOD, while able to bind either Fe or Mn, is only active with one. Thus, the structure and activity of the enzyme reflect interactions between the protein and the metal ion, and these interactions are metal ion specific. The goal of the research is (1) to determine how much of the protein is affected by binding of small molecules to the metal ion, and (2) to describe the protein structural change in terms of relative movement of the modules that make up the protein structure. The work will also determine why MnSOD protein is not active with Fe bound and FeSOD protein is not active with Mn bound. This new understanding of the protein-metal cooperation that is necessary for metalloenzyme catalytic activity will advance ongoing efforts to design or modify metalloenzymes to catalyze useful reactions in drug synthesis, waste detoxification and chemical sensors. ***

9418181米勒该研究将采用含铁和锰的超氧化物歧化酶（SOD）作为模型系统，并利用核磁共振光谱（NMR）的独特能力直接观察蛋白质基团并监测溶液中的结构。 目的是阐明金属离子位点和蛋白质的SOD底物类似物结合的金属离子的构象之间的相互作用，并确定为什么FeSOD蛋白是无活性的与锰结合而不是铁，和MnSOD蛋白是无活性的与铁结合而不是锰。 同位素编辑的NMR将与氨基酸特异性同位素标记结合使用，以产生仅显示标记氨基酸信号的大幅简化的光谱。 有和没有底物类似物叠氮化物结合的SOD光谱的比较将识别受叠氮化物结合影响的氨基酸，并指示蛋白质在金属离子处对事件的构象响应的机制。 通过监测结构域或亚基之间界面处的氨基酸，将有可能确定构象变化是否涉及构象变化的潜在一般机制，包括（1）由金属离子桥接的结构域的相对运动，（2）亚基的相对运动和（3）通过氢键网络传播构象效应。 这些研究建立在SOD已知结构的基础上，并利用NMR直接观察质子的独特能力，提供关于它们参与氢键的信息，并提供定性和定量分析。 关于蛋白质在不受晶体堆积约束的溶液中的结构和动力学的信息。 这项工作还将确定为什么含有错误金属离子的SOD是无活性的，通过评估它们对催化的各个元素的能力，包括每个半反应，还原中点电位，质子捐赠，底物类似物结合和活性位点结构，从而指定催化无能的化学原因。 将使用定点和随机诱变将MnSOD蛋白转化为FeSOD。 这些SOD的表征将揭示哪些氨基酸变化是重要的，这些氨基酸变化支持与错误金属的活性的程度，以及与正确金属的活性的代价。 金属酶联合收割机将金属中心的反应性与酶的精确特异性和控制结合在一起。 含铁超氧化物歧化酶（FeSODs）和同源的含锰超氧化物歧化酶（MnSODs）是催化超氧化物转化为分子氧和过氧化氢的金属酶。 SOD相对较小，非常稳定和可溶，但也体现了金属酶催化的两个核心特征：金属位点和蛋白质被认为会影响彼此的结构，并且每种类型的SOD虽然能够结合Fe或Mn，但只对其中一种有活性。 因此，酶的结构和活性反映了蛋白质和金属离子之间的相互作用，并且这些相互作用是金属离子特异性的。 该研究的目标是（1）确定小分子与金属离子结合对蛋白质的影响程度，以及（2）根据组成蛋白质结构的模块的相对运动来描述蛋白质结构变化。 本工作还将确定为什么MnSOD蛋白与Fe结合时不具有活性，而FeSOD蛋白与Mn结合时不具有活性。 这种对金属酶催化活性所必需的蛋白质-金属合作的新理解将推动正在进行的设计或修饰金属酶的努力，以催化药物合成，废物解毒和化学传感器中的有用反应。 ***