Structure/Function of Mn and Fe Superoxide Dismutases and Related Enzymes

描述（由申请人提供）：需氧生物体利用分子氧进行各种苛刻的氧化转化，如半胱氨酸双加氧酶（CDO）催化的半胱氨酸转化为半胱氨酸亚磺酸。然而，有氧代谢也会导致各种副反应，产生活性氧，如超氧阴离子（O2-）。因此，自然界开发了一种有效的除氧策略，涉及称为超氧化物歧化酶（SOD）的金属酶，其需要Fe，Mn，Cu/Zn或Ni金属辅因子的催化活性。在所有好氧生物中发现，SOD使超氧自由基与O2和H2 O2不成比例。该提案中概述的研究的长期目标是：7确定Fe和MnSOD的关键几何和电子结构特征，这些特征有助于这些酶的高催化速率。7从分子水平上深入了解SOD和CDO的反应机理。7利用我们的知识将新的酶功能工程化到FeSOD中。基于这些目标，我们制定了以下具体目标：1.阐明了铁和锰超氧化物歧化酶活性中心性质的远程调谐机制。2.探索所谓的变形SOD克服提供一个活性位点环境，容忍Fe和Mn支持的活动的挑战的手段。3.获得分子水平的洞察力的Fe-和MnSOD的催化机制。4.确定镍支持的SOD活性的关键结构要素。5.研究CDO的结构/功能关系和催化机制。6.将CDO活性设计为FeSOD。为了实现这些目标，我们将使用相结合的光谱/计算方法来研究天然酶和选定突变体的静息态和底物（类似物）复合物。Fe和MnSOD为研究活性位点性质的远程调节机制提供了几乎理想的蛋白质支架，例如金属离子还原电位和底物（类似物）/活性位点相互作用。通过将我们的研究扩展到NiSOD和CDO，我们可以测试和完善我们的假设，即外层氨基酸残基有助于优化金属酶活性位点的原则。从实践的角度来看，在我们提出的研究中获得的见解可能为合理设计SOD和CDO模拟物用于药物应用提供基础，例如治疗阿尔茨海默病和帕金森病。超氧自由基阴离子和游离半胱氨酸都已被证明在几种神经退行性疾病中起作用，包括运动神经元疾病、帕金森病和阿尔茨海默病。在正常情况下，这些物质的浓度通过超氧化物歧化酶和半胱氨酸双加氧酶维持在非常低的水平，这是本提案的重点。在我们提出的研究中获得的见解可能为合理设计用于药物应用的酶模拟物提供合适的基础，例如治疗神经退行性疾病。

DESCRIPTION (provided by applicant): Molecular oxygen is utilized by aerobic organisms to perform a variety of demanding oxidative transformations, such as the conversion of cysteine to cysteine sulfinic acid catalyzed by cysteine dioxygenase (CDO). However, aerobic metabolism also leads to various side reactions that produce reactive oxygen species, such as the superoxide anion (O2-). Nature has therefore developed an effective strategy for O2- removal that involves metalloenzymes known as superoxide dismutases (SODs), which require either Fe, Mn, Cu/Zn, or Ni metal cofactors for catalytic activity. Found in all aerobic organisms, SODs disproportionate the superoxide radical to O2 and H2O2. The Long-Term Objectives of the research outlined in this proposal are: 7 To identify key geometric and electronic structural features of the Fe- and MnSODs that contribute to the high catalytic rates of these enzymes. 7 To obtain molecular level insight into the reaction mechanisms of SODs and CDO. 7 To utilize our knowledge for engineering novel enzymatic functions into FeSOD. With these goals in mind, we have formulated the following Specific Aims: 1. Elucidate the mechanism of long-range tuning of the active site properties in Fe- and MnSODs. 2. Explore the means by which the so-called cambialistic SODs overcome the challenge of providing an active-site environment that tolerates both Fe- and Mn-supported activity. 3. Obtain molecular-level insight into the catalytic mechanisms of Fe- and MnSODs. 4. Identify key structural elements for Ni-supported SOD activity. 5. Investigate structure/function relationships and the catalytic mechanism of CDO. 6. Engineer CDO activity into FeSOD. To accomplish these goals, we will use a combined spectroscopic/computational approach for studying the resting states and substrate (analog) complexes of the native enzymes and selected mutants. The Fe- and MnSODs provide almost ideal protein scaffolds for investigating the mechanisms of long- range tuning of active-site properties, such as the metal ion reduction potential and substrate (analog)/active site interactions. By extending our studies to NiSOD and CDO, we can test and refine our hypotheses regarding the principles by which outer-sphere amino acid residues contribute to the optimization of metalloenzyme active sites. From a practical point of view, insights gained in our proposed studies may provide the basis for the rational design of SOD and CDO mimics for pharmaceutical applications, such as the treatment of Alzheimer disease and Parkinson disease. Both the superoxide radical anion and free cysteine have been shown to play a role in several neurodegenerative diseases, including motor neuron disease, Parkinson disease, and Alzheimer disease. Under normal circumstances, the concentration of these species is maintained at very low levels by superoxide dismutases and cysteine dioxygenase, which are the focus of this proposal. Insights gained in our proposed studies may provide a suitable basis for the rational design of enzyme mimics for pharmaceutical applications, such as the treatment of neurodegenerative diseases.