Enzyme Mis-Metallation, Consequences and Opportunities

好氧生物利用氧气从氧化中提取大量能量 食物的新陈代谢，并对一些重要的化学物质进行氧化反应 涉及抗生素生物合成、外源物质代谢的转化， 构建生物聚合物，以及更多。为了获得这种化学反应，O2必须是 被激活，这通常是通过含铁的酶完成的。游离态活性O2 与细胞的许多成分反应，随之而来的损害导致许多 疾病包括糖尿病、关节炎、神经退行性疾病、癌症和 衰老的症状。针对这一系列疾病的一个关键的生化防御是 酶超氧化物歧化酶(SOD)，催化亲本的转化 活化的O2物种，超氧化物，转化为O2+H2O2。目前的工作重点是 大肠杆菌超氧化物歧化酶，进化为使用铁作为其催化金属离子(FeSOD)，具有 一种改用锰的同源酵母超氧化物歧化酶的体内实验 (MnSODSC)。酵母酶与人类线粒体高度同源 因此，它是人类酶的模型，同时也借鉴了 酵母的广泛的遗传和生理研究，这是不能在 人类。主要的推进体解决了FeSOD的铁通过(和通过 O2-激活酶的铁)与激活的O2相互作用，而不需要 屈服于它，以及FeSOD活性中心调节Fe的反应活性的途径 (这样它就会停用超氧化物)。酵母菌超氧化物歧化酶的初步研究 通过酵母将铁替代为人的超氧化物歧化酶的可能的病理后果 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。这个 主要推动力直接建立在申请人实验室过去的研究基础上，使用的是E.ColiFeSOD 已经证明被困在所使用的两个状态之一的变体 FeSOD的催化循环，但保留了其他类似天然的活性部位。因为他们 不能在催化循环中进行，它们是生成 酶-底物和酶-产物复合体的模型，了解FeSOD是如何 活性中心与其底物和产物结合并相互作用。由熟练人员进行的详细研究 光谱学家以及力学和热力学研究将提供一种 异常完整的超氧化物歧化酶周转中间体模型图，以及 对铁酶如何处理其基本但危险的底物的见解。 已建立的酶学和生物物理方法将与停用的- 流动和冷冻淬火方法，以扩大实验室治疗短命的能力 复合体。酵母系统将启动。分子间反应的初步研究 过氧化氢和铁取代的锰超氧化物歧化酶将评估铁的潜在意义 置换成超氧化物歧化酶，对人体产生氧化应激。在大肠杆菌中产生的突变和 作为机械性工作的一部分，还将测试扭转 铁替代酵母MnSOD可能的毒性效应，从而进行翻译 对潜在治疗方法的机械洞察力。

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.