NOVEL SUBSTRATE OXIDATION BY ENZYME ENGINEERING

描述：（改编自申请人的摘要）该提案扩展了 利用细胞色素c的氧铁基血红素中心的前期研究 过氧化物酶（CCP）用于氧化新的小分子底物。 以前的工作集中在蛋白质环境如何定义物理， 血红素过氧化物酶光谱和功能性质。 这些 表明，由某些血红素酶催化的氧化反应可能是 被招募到别人的绞刑架上 取得了重大进展 在上一个资助期， 通过空腔互补将位点插入酶中。 这些结果说明 这种方法用于将潜在的衬底放置在 血红素活性位点，此外，它们还为 回答一些关于弱相互作用的更一般的问题， 配体-蛋白质复合物和表面环运动， 预期。 在下一个供资周期，首席研究员力求 完善和扩展这些概念，以提高使用 之前获得的关于蛋白质因子的信息 环境，控制的能量，反应性，和特异性， 活性位点，以便将新的底物结合引入酶中 并描述这些相互作用及其潜在的 被血红素氧化 将提出两条一般性的调查路线， 下节课- 1）这些人工结合位点能告诉我们什么关于血红素的信息 酶功能和蛋白质-配体相互作用，以及2）这些位点可以 用来改造具有新功能的酶 更具体地 第一个领域，首席研究员将探索更一般的方面 在CCP中创建人工腔并表征特异性， 与小分子结合到这些分子上的动力学和能量学 网站. 第二个重点将利用这项工作来审查 氧化化学可能发生在不同的位置， 位置相对于heme。 这些研究可能有助于提供一个 更多地了解是什么因素控制着 一方面，过氧化物酶表现出的活性， 电子或氢原子转移，另一方面是单加氧酶， 通过铁基氧代转移化学进行操作。 因此，这些人工酶 可能有助于更好地了解P450、一氧化氮 氧化物合酶、吲哚胺2，3-双加氧酶和前列腺素合酶， 每一种都具有重要的医学意义。

DESCRIPTION: (Adapted from applicant's abstract) The proposal extends previous studies on utilizing the oxyferryl heme center of cytochrome c peroxidase (CCP) for the oxidation of novel small molecule substrates. Previous work focused on how the protein environment defines the physical, spectroscopic and functional properties of heme peroxidases. These suggested that oxidative reactions catalyzed by some heme enzymes might be recruited into the scaffold of others. Significant progress has been made in the last grant period on efforts to introduce small molecule binding sites into the enzyme by cavity complementation. These results illustrate the feasibility of this approach for placing potential substrates near the heme active site and in addition, they have provided a framework for answering some more general questions about weak interactions in ligand-protein complexes and surface loop movements that were not originally anticipated. In the next funding cycle the Principal Investigator seeks to refine and extend these concepts to focus to an increasing degree on using the information obtained earlier about the factors of the protein environment that control the energetics, reactivity, and specificity of the active site, in order to introduce novel substrate binding into the enzyme and to characterize these interactions and their potential for being oxidized by the heme. Two general lines of inquiry will be proposed for the next period- 1) what can these artificial binding sites tell us about heme enzyme function and protein-ligand interactions, and 2) can these sites be used to engineer enzymes with novel function? More specifically, in the first area, the Principal Investigator will explore the more general aspects of creating artificial cavities in CCP and characterizing the specificity, kinetics and energetics associated with small molecule binding to these sites. The second emphasis will draw upon this work to examine the oxidative chemistry that may occur at sites that are placed at different positions with respect to the heme. These studies may help provide an increased understanding of what factors control the very different activities exhibited by peroxidases on one hand, which oxidize substrates by electron or hydrogen atom transfer, and monooxygenases on the other, which operate by ferryl oxo-transfer chemistry. Thus, these artificial enzymes may help provide a better understanding of the function of P450, nitric oxide synthase, indoleamine 2,3-dioxygenase, and prostaglandin synthase, each of which have significant medical importance.