NOVEL SUBSTRATE OXIDATION BY ENZYME ENGINEERING

拟议的研究将探讨利用 细胞色素c过氧化物酶（CCP）的氧化亚铁血红素中心 新型小分子底物。 将采取两种办法， 发展这个概念：建设一些人工变异 和杂合酶，其可赋予对多种 小分子，并详细调查的结构和 这些和其他改变对血红素的生物物理影响 活性部位 这样的人工酶将与这样的酶相关。 生物学上重要的催化剂如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 依赖性木质素酶 在这些研究中，一个核心的和未解决的 问题将得到解决：蛋白质环境如何指导 氧化血红素的化学反应，如烯烃 环氧化和羟基化，分子内电子转移， 自由基化学和中间金属离子氧化？ 以下 具体目标将被解决：1）蛋白质的作用， 环境在控制CCP的氧化还原性能方面的作用将是 考察 2)研究了不同的离子对血红素电子结构的影响， 调节金属-配体相互作用和配位状态将是 研究了 3)Trp-191自由基和替代物的相互作用 将探索血红素的自由基中心。 4)人工基质 位点将被引入到近端和远端血红素环境中， 并且将检查这些蛋白质的新底物氧化。 第五章） 部分其他过氧化物酶将被嫁接到CCP序列中 为了将宽范围的底物氧化能力引入其中， 结构 5)这些酶中的每一种都将在结构上表征 以指导进一步的设计工作。 反应性的变化，金属- 血红素的配体结合、电子转移效率和氧化还原电位 酶是由于部分敏感的金属电子 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变化， 目前还很难预测这样一个 结构的变化将对金属电子能级产生影响， 从而影响蛋白质的功能。 由于这些原因， 不仅要研究这些酶的结构变化， 功能后果，但要衡量并直接联系随之而来的后果 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.