Enzyme Mis-Metallation, Consequences and Opportunities

好氧生物利用O2从氧化过程中提取大量能量， 代谢的食物，并利用氧化反应的一些重要的化学品 涉及抗生素生物合成的转化，外源性物质的代谢， 生物聚合物的构建等等。为了获得这种化学物质，O2必须是 激活，这通常是通过含铁的酶来实现的。游离活性O2 与细胞的许多成分发生反应，并对许多细胞造成损害。 疾病，包括糖尿病、关节炎、神经退行性疾病、癌症和 老年的症状。一个关键的生化防御这一系列的疾病是 酶超氧化物歧化酶（SOD），其催化母体转化 活性O2物种，超氧化物，O2 + H2 O2。目前的工作重点是 大肠杆菌SOD进化为使用Fe作为其催化金属离子（FeSOD）， 支持使用Mn代替的同源酵母SOD的体内实验 （MnSODSc）。酵母酶与人类线粒体高度同源 MnSOD，因此可以作为人体酶的模型，同时也借鉴了 酵母的广泛遗传和生理研究，这不能在 人类主要的推力解决机制，其中铁的FeSOD（和 O2-活化酶的Fe）与活化的O2相互作用， 屈服于它，以及FeSOD的活性位点调节Fe的反应性的方法 (such它将使超氧化物失活）。酵母SOD的研究启动了对SOD的研究 通过酵母将Fes取代为人类SOD可能的病理后果 模型，并测试一种突变，通过这种突变可以预防或纠正这种病理。的 主要推力直接建立在申请人实验室过去的研究基础上，使用E。大肠杆菌FeSOD 已被证明被困在两个国家之一所采用的变体， FeSOD的催化循环，但保留否则天然样的活性位点。因为他们 不能通过催化循环，它们是理想的系统，在其中产生 酶-底物和酶-产物复合物的模型，以了解FeSOD 活性位点与其底物和产物结合并相互作用。技术人员的详细研究 光谱学家，以及机械和热力学研究将提供一个 非常完整的SOD周转中间体模型，以及 深入了解铁酶如何处理其重要但危险的底物。 已建立的酶学和生物物理学方法将与停止- 流动和冷冻淬火的方法，以扩大实验室的能力， 配合物将启动酵母系统。初步研究了 过氧化物和铁取代的MnSOD将评估铁的潜在意义 取代成SOD，对人体氧化应激。在E.杆菌和 被描述为机械工作的一部分，也将被测试的能力，扭转 可能的毒性作用的铁替代酵母MnSOD，从而进行翻译 对潜在治疗方法的机械见解。

Aerobic organisms exploit O2 to extract large amounts of energy from oxidative metabolism of food, and employ oxidative reactions for a number of important chemical transformations involved in antibiotic biosynthesis, metabolism of xenobiotics, construction of biopolymers, and more. In order to access this chemistry, O2 must be activated, and this is often accomplished via Fe-containing enzymes. Free activated O2 reacts with many components of cells and the damage that ensues contributes to many diseases including diabetes, arthritis, neurodegenerative conditions, cancer, and the symptoms of old age. A crucial biochemical defense against this array of ills is the enzyme superoxide dismutase (SOD), which catalyzes conversion of the parent activated O2 species, superoxide, to O2 + H2O2. The current work focuses on the Escherichia coli SOD that is evolved to use Fe as its catalytic metal ion (FeSOD), with supporting in-vivo experiments on a homologous yeast SOD that employs Mn instead (MnSODSc). The yeast enzyme is highly homologous to the human mitochondrial MnSOD, and thus serves as a model for the human enzyme, while also drawing on extensive genetic and physiological studies of yeast, which cannot be performed on humans. The major thrusts address mechanisms by which the Fe of FeSOD (and by extension the Fe of O2- activation enzymes) interacts with activated O2 without succumbing to it, and means by which the active site of FeSOD tunes the reactivity of Fe (such that it will de-activate superoxide). The efforts on yeast SOD initiate investigations of possible pathological consequences of Fesubstitution into human SOD, via the yeast model, and test a mutation by which this pathology could be prevented or corrected. The major thrust builds directly on past research in the applicant's lab, using E. coli FeSOD variants that have been shown to be trapped in one of the two states employed by FeSOD's catalytic cycle, but to retain otherwise native-like active sites. Because they cannot progress through the catalytic cycle, they are ideal systems in which to generate models of enzyme-substrate and enzyme-product complexes, to learn how the FeSOD active site binds and interacts with its substrate and products. Detailed studies by skilled spectroscopists, as well as mechanistic and thermodynamic studies will provide an exceptionally complete picture of models of the intermediates of SOD turnover, and insights into how Fe enzymes handle their essential but dangerous substrate. Established enzymological and biophysical approaches will be blended with stopped- flow and freeze-quenched methods to extend the lab's ability to treat short-lived complexes. The yeast system will be launched. Initial studies of the reactions between peroxide and Fe-substituted MnSOD will evaluate the potential significance of Fe substitution into SOD, to human oxidative stress. A mutation developed in E. coli and characterized as part of the mechanistic work will also be tested for ability to reverse the possible toxic effects of Fe substitution into yeast MnSOD, thus carrying translating mechanistic insights into potential treatments.