SPECTROSCOPIC STUDIES OF PYRUVATE FORMATE LYASE ACTIVASE

这个项目的目的是从机械的角度来理解 酶、铁和S-腺苷蛋氨酸在反应中的作用 由丙酮酸甲酸裂解酶激活酶(AE)催化。这个 丙酮酸甲酸裂解酶激活酶利用铁和SAM生成 丙酮酸甲酸裂解酶(PFL)上稳定的甘氨酸基，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，几乎没有 了解金属的配位环境或铁的作用 在激进的一代。正如提案中所描述的，AE似乎是一种 一种新兴的酶，所有这些酶都利用铁和SAM来 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催化的反应- 依赖的酶，因此出现了关于潜在的问题 腺苷钴胺和铁/SAM介导的机制相似性 酶促反应。本提案中所述的初步研究 证明Ae，就像其他成员一样， 酶，含有铁-硫团簇。这篇文章中描述的工作 Proposal的具体目标是表征声发射金属 中心及金属中心和自组装膜在细胞周期中的作用 在PFL上产生一个激进分子。在更广泛的意义上，这些研究将 也导致了对这一新的机械论的发展理解 铁和依赖SAM的酶的一类。《公约》的具体目标 建议书如下： 1.通过以下方式确定最大比活动量的要求 对表达系统和表达条件进行了研究。 2.丙酮酸甲酸裂解酶铁硫簇的表征 激活酶(AE)。该方法将包括对本地人的检查 用UV-Vis、MCD、EPR、拉曼和穆斯堡尔谱进行了声发射。 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)在土壤中的作用问题 酶分析将使用光谱和动力学两种方法。 接近了。 4.探讨声发射铁硫中心的生物相关性 使用动力学方法包括：1.酶促反应的相关性 活性与铁和硫化物的含量，以及2，详细检查 当一个完整的铁硫时，对酶活性的要求 中心在现场。 5.利用金属生色团作为光谱探针用于 声发射与SAM和PFL的相互作用。声发射的铁硫中心将 提供了一个方便的光谱手柄，用于监测 底物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.