NOVEL SUBSTRATE OXIDATION BY ENZYME ENGINEERING

拟议的研究将探索利用 细胞色素c过氧化物酶(CCP)的氧铁血红素中心的氧化作用 新的小分子底物。将采取两种方法来 发展这个概念：构建一些人工变异 和混合酶，可以赋予一个品种氧化活性 以及对其结构和结构的详细研究。 这些变化和其他变化对血红素的生物物理影响 活动站点。这样的人造酶将与这样的 重要的生物催化剂，如P450，一氧化氮合酶， 吲哚胺2，3-双加氧酶、辣根过氧化物酶和锰 依赖木质素酶。在这些研究中，一个中心和悬而未决的问题 问题将被解决：蛋白质环境如何引导 氧化后的亚铁血红素发生不同的化学反应，如烯烃 环氧化和羟基化，分子内电子转移和 自由基化学和中间金属离子氧化？以下是 具体目标将被解决：1)蛋白质的作用 环境在控制CCP的氧化还原性质中的作用将是 检查过了。2)对血红素电子结构的影响 调节金属-配体的相互作用和配位状态将是 学习。3)Trp-191自由基及其替代物的相互作用 我们将探索以亚铁血红素为中心的自由基。4)人造底物 地点将被引入到近端和远端的血红素环境中， 这些蛋白质将被检测为新的底物氧化。5) 其他部分过氧化物酶将被嫁接到CCP序列中 将广泛的底物氧化能力引入到这个 结构。5)这些酶中的每一种都将被结构表征 以指导进一步的设计工作。反应性的变化，金属- 血红素的配基结合、电子转移效率和氧化还原电位 酶的部分原因是金属电子的敏感性。 价态对共价性和化学环境的微妙影响。 然而，即使已经引入了明确的结构性变化， 到目前为止，还很难预测这样一种 结构的变化会对金属的电子能级和 从而对蛋白质的功能产生影响。出于这些原因，它是重要的 不仅要研究这些酶的结构变化和它们的 功能后果，但要衡量和直接联系随后的 金属电子结构性质的变化。

The proposed studies will explore the potential of utilizing the oxyferryl heme center of cytochrome c peroxidase (CCP) for the oxidation of novel small molecule substrates. Two approaches will be taken to develop this concept: The construction a number of artificial variants and hybrid enzymes that may confer oxidizing activity toward a variety of small molecules, and a detailed investigation of the structural and biophysical effects that these and other alterations have upon the heme active site. Such artificial enzymes will have relevance to such biologically important catalysts as P450, nitric oxide synthase, indoleamine 2,3-dioxygenase, horseradish peroxidase, and manganese dependent ligninase. Throughout these studies, a central and unresolved question will be addressed: How does the protein environment direct the chemistry of the oxidized heme toward such diverse reactions as alkene epoxidation and hydroxylation, intramolecular electron transfer and radical chemistry, and intermediary metal ion oxidation? The following specific aims will be addressed: 1) The role that the protein environment plays in controlling the redox properties of CCP will be examined. 2) The effects on the heme electronic structure resulting from modulating the metal-ligand interaction and coordination state will be studied. 3) the interaction of the Trp-191 free radical and alternative radical centers to the heme will be explored. 4) Artificial substrate sites will be introduced into the proximal and distal heme environment, and these proteins will be examined for novel substrate oxidation. 5) Portions of other peroxidases will be grafted into the sequence of CCP to introduce a broad range of substrate oxidizing capabilities into this structure. 5) Each of these enzymes will be structurally characterized to guide further design efforts. The variations in reactivity, metal- ligand binding, electron transfer efficiency, and redox potential of heme enzymes are due in part to the sensitivity of the metal electronic valence states to subtle effects of covalency and chemical environment. However, even if a well defined structural change has been introduced, it is very difficult as yet to predict the consequences that such a change in structure will have on the metal electronic energy levels and thus on the function of the protein. For these reasons it is important to study not just the structural alteration of these enzymes and their functional consequences, but to measure and relate directly the ensuing changes in the properties of the metal electronic structure.