SPECTROSCOPIC STUDIES OF PYRUVATE FORMATE LYASE ACTIVASE

这个项目的目的是获得一个机械的理解， 酶、铁和S-腺苷甲硫氨酸（SAM）在反应中的作用 由丙酮酸甲酸裂解酶激活酶（AE）催化。 的 丙酮酸甲酸裂解酶激活酶利用铁和SAM产生 丙酮酸甲酸裂解酶（PFL）上的稳定甘氨酰自由基，从而 激活它以实现正常的生物功能 而科扎里奇和 同事们已经证明了Fe（II）与AE的化学计量结合， 已知金属配位环境或铁的作用 在激进的一代。 如提案所述，AE似乎是一个 一种新兴的酶，所有这些酶都利用铁和SAM， 产生自由基或催化自由基介导的反应。 在许多情况下 这些反应类似于腺苷钴胺素催化的那些反应， 依赖酶，因此出现了关于潜在的 腺苷钴胺素和铁/SAM介导的 酶反应。 本提案所述的初步研究 表明AE，像其他成员的ht铁/SAM类的 酶，含有铁硫簇。 本文所描述的工作 该提案的具体目标是表征AE金属 中心和金属中心和SAM的作用的阐明， 在PFL上生成自由基。 在更一般的意义上，这些研究将 也导致了对这种新的机械理解的发展， 一类铁和SAM依赖酶。 的具体目标 建议如下： 1. 通过以下方法确定最大比活度的要求： 对表达系统和表达条件进行了研究。 2. 丙酮酸甲酸裂解酶中铁硫簇的表征 活化酶（AE）。 该方法将包括检查本地 AE与UV-Vis，MCD，EPR，拉曼和穆斯堡尔光谱。 3. 研究Fe（II）在团簇转化和/或AE中的作用 催化活性 添加的Fe（II）在 酶的测定将使用光谱和动力学 接近。 4. 探讨AE铁硫中心的生物学意义 使用动力学方法，包括：1.酶的相关性 活性与铁和硫化物的含量，2，详细检查 当一个完整的铁-硫 中心在。 5. 利用金属生色团作为光谱探针， 声发射与SAM和PFL相互作用。 AE的铁硫中心将 提供了一个方便的光谱处理监测的变化， 基质结合时的金属配位环境。

The aim of this project is to obtain a mechanistic understanding of the roles of the enzyme, iron and s-adenosylmethionine (SAM) in the reaction catalyzed by the pyruvate formate-lyase activating enzyme (AE). The pyruvate formate-lyase activating enzyme utilizes iron and SAM to generate a stable glycyl radical on pyruvate formate-lyase (PFL), thereby activating it for normal biological function. While kozarich and coworkers have demonstrated stoichiometric binding of Fe(II) to AE, little is known about the metal coordination environment or the role of the iron in radical generation. As described in the proposal, AE appears to be one of an emerging class of enzymes, all of which utilize iron and SAM to generate radicals or catalyze radical-mediated reactions. In many cases these reactions are analogous to those catalyzed by adenosylcobalamin- dependent enzymes, and thus questions have arisen regarding the potential mechanistic similarities between adenosylcobalamin and iron/SAM-mediated enzymatic reactions. preliminary studies described in this proposal demonstrate that AE, like the other members of ht iron/SAM class of enzymes, contains an iron-sulfur cluster. The work described in this proposal has as its specific goal the characterization of the AE metal center and the elucidation of the role of the metal center and SAM in generating a radical on PFL. in a more general sense these studies will also lead to the development of a mechanistic understanding of this new class of iron and SAM-dependent enzymes. The specific aims of the proposal are as follows: 1. To determine the requirements for maximal specific activity by investigating the expression system and the expression conditions. 2. To characterize the iron-sulfur cluster in pyruvate formate-lyase activating enzyme (AE). The approach will include examination of native AE with UV-Vis, MCD, EPR, Raman, and Mossbauer spectroscopy. 3. To examine the role of Fe(II) in cluster conversion and/or in AE catalytic activity. The question of the role of the added Fe(II) in the enzyme assay will be addressed using both spectroscopic and kinetic approaches. 4. To probe the biological relevance of the iron-sulfur center of AE using kinetic approaches including: 1. the correlation of enzymatic activity with the iron and sulfide content, and 2, a detailed examination of the requirements for enzymatic activity when an intact iron-sulfur center is present. 5. To utilize the metal chromophore as a spectroscopic probe for the interaction of AE with SAM and PFL. The iron-sulfur center of AE will provide a convenient spectroscopic handle for monitoring alterations in the metal coordination environment upon substrate binding.