NOVEL SUBSTRATE OXIDATION BY ENZYME ENGINEERING

描述：(改编自申请人的摘要)提案扩展 利用细胞色素c的氧铁血红素中心的研究进展 用于新型小分子底物氧化的过氧化物酶(CCP)。 以前的工作集中在蛋白质环境如何定义物理， 血红素过氧化物酶的光谱和功能性质。这些 提示某些血红素酶催化的氧化反应可能是 被招募到其他人的绞刑架上。已经取得了重大进展 在上一次的资助期内，努力引入小分子结合 以空穴互补的方式进入酶中。这些结果说明 这种方法将潜在的衬底放置在 此外，它们还提供了一个框架， 回答有关弱相互作用的一些更一般的问题 配体-蛋白质复合体和表面环运动 已经预料到了。在下一个供资周期中，首席调查员寻求 提炼和扩展这些概念，以便更多地关注使用 早先获得的有关蛋白质因素的信息 控制能量、反应性和专一性的环境 活性部位，以便在酶中引入新的底物结合 并描述这些相互作用及其存在的可能性 被亚铁血红素氧化的。将提出两条大致的调查路线 下一期--这些人工结合位点能告诉我们关于血红素的什么信息？ 酶功能和蛋白质-配体相互作用，以及2)这些位点是否可以 用于设计具有新功能的酶？更具体地说，在 第一个领域，首席调查员将探索更一般的方面 在CCP中制造人工空洞并表征其特异性， 与小分子结合有关的动力学和能量学 网站。第二个重点将利用这项工作来审查 可能发生在不同位置的氧化化学 相对于亚铁血红素的位置。这些研究可能有助于提供一种 加深了对控制差异的因素的理解 一方面，过氧化物酶表现出的活性通过以下方式氧化底物 电子或氢原子转移，以及另一端的单加氧酶，这 通过铁氧基转移化学进行操作。因此，这些人造酶 可能有助于更好地了解P450、NO的功能 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.