STRUCTURAL BIOLOGY OF PEROXIDATION BY PGH SYNTHASES

我们持续暴露在氧自由基和相关的氧化剂中， 与衰老相关的慢性和急性疾病状态（例如， 动脉粥样硬化、关节炎和癌症）。前列腺素H合酶（PGHS）-1 和-2是将花生四烯酸转化为血红素的完整膜酶 前列腺素级联反应的前体。PGHS亚型 产生自由基，破坏细胞和细胞成分并激活 环境污染物。PGHS过氧化物酶构成了一个独特的， 该结构的互补途径与 氧化损伤有两个原因。首先，PGHS的三级结构是 不仅与其他哺乳动物过氧化物酶同源， 哺乳动物过氧化物酶大家族。第二，理解 酶的结构是理解酶的作用机制的先决条件， 生成氧化产物。 酶-配体相互作用的性质和结构/功能 在天然PGHS过氧化物酶的关系，以及在定点 突变体将使用停流紫外-可见吸收进行探索 光谱学、共振拉曼光谱学和X射线晶体学。的 具体目标如下。首先，我们将研究 酶-配体相互作用和相关的结构-功能关系 在PGHS同种型的天然过氧化物酶中表征血红素铁 环境通过使用血红素铁配体，并确定，然后 使用弱（即， 更稳定的）还原剂如2-氨基芴和苄基异羟肟 酸第二，我们将研究结构/功能关系， 绵羊PGHS-1过氧化物酶通过突变分析的过氧化物酶网站。 第三，我们将研究人体内的结构-功能关系， PGHS-2过氧化物酶，通过检查具有等同突变的修饰PGHS-2 如绵羊PGHS-1。最后，我们将尝试遵循结构 参与自由基生成的变化，以阐明 自由基转移和过氧化物酶依赖性失活。目标是 识别和稳定长效氧化或自由基状态 由过氧化物酶反应产生，在溶液和晶体中。一 另一个目的是确定自由基损伤的性质 导致PGHS过氧化物酶失活， 在其他生物系统中蛋白质的自由基损伤。

Our constant exposure to oxygen radicals and related oxidants can initiate chronic and acute disease states associated with aging (e.g., atherosclerosis, arthritis, and cancer). Prostaglandin H synthase (PGHS)-1 and -2 are integral membrane heme-enzymes which convert arachidonic acid into the precursors of the prostaglandin cascade. The PGHS isoforms generate free radicals that damage cell and cell components and activate environmental contaminants. The PGHS peroxidase constitutes a distinct and complementary pathway to the structure is relevant to pathology of oxidative damage for two reasons. First, the tertiary structure of PGHS is not only homologous to that of other mammalian peroxidases but also to the large family of mammalian peroxidases. Second, an understanding of the enzyme's structure is a prerequisite for understanding the mechanism that generates oxidative products. The nature of enzyme-ligand interactions and structure/function relationships in the native PGHS peroxidases, as well as in site-directed mutants will be explored using stopped flow UV-Vis absorption spectroscopy, resonance Raman spectroscopy and X-ray crystallography. The Specific Aims are as follows. First, we will investigate the nature of enzyme-ligand interactions and associated structure-function relationships in the native peroxidase of PGHS isoforms to characterize the heme iron environment through the use of heme iron ligands and to identify and then characterize the binding site(s) for reducing substrates using weak (i.e., more stable) reductants such as 2-aminofluorene and benzyl hydroxamic acid. Second, we will study the structure/function relationships in the ovine PGHS-1 peroxidase via mutational analyses in the peroxidase site. Third, we will study the structure-function relationships in the human PGHS-2 peroxidase by examining modified PGHS-2 with equivalent mutations as in ovine PGHS-1. Finally, we will attempt to follow the structural changes involved in free radical generation to elucidate the mechanism of free radical transfer and peroxidase-dependent inactivation. The goal is to identify and stabilize long-lived oxidative or free radical states created by the peroxidase reaction, in solution and in the crystal. A subsidiary goal is also to determine the nature of the free radical damage that leads to inactivation of the PGHS peroxidase and to develop a model for free radical damage of protein in other biological systems.