STRUCTURAL BIOLOGY OF PEROXIDATION BY PGH SYNTHASES

我们持续暴露在氧自由基和相关氧化剂中会引发 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合成酶(PGHS)-1 和-2是转化花生四烯酸的完整的膜血红素酶 变成前列腺素瀑布的前体。PGHS亚型 产生破坏细胞和细胞成分的自由基，并激活 环境污染物。PGHS过氧化物酶构成了一种独特的 结构的互补途径与肿瘤的病理机制有关 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的过氧化物酶同源，而且与 哺乳动物过氧化物酶的大家族。第二，对 酶的结构是理解这一机制的先决条件 产生氧化产物。 酶-配体相互作用的性质和结构/功能 天然PGHS过氧化物酶以及位点定向酶之间的关系 将使用停止流动的UV-Vis吸收来探索突变体 光谱学、共振拉曼光谱和X射线结晶学。这个 具体目标如下。首先，我们将调查这一事件的性质 酶-配体相互作用及其相关结构-功能关系 在天然过氧化物酶的PGHS异构体中表征血红素铁 通过使用环境中的血红素铁配体进行鉴定，然后 表征用于还原底物的弱(即， 更稳定的)还原剂，如2-氨基荧烯和苯基异羟胺 酸。第二，我们将研究结构/功能关系在 绵羊PGHS-1过氧化物酶的酶切位点突变分析。 第三，我们将研究人类的结构-功能关系 用等量突变检测修饰的PGHS-2过氧化物酶 就像绵羊PGHS-1一样。最后，我们将尝试遵循结构 参与自由基产生的变化，以阐明其机制 自由基转移和过氧化物酶依赖的失活。我们的目标是 识别和稳定长寿命的氧化或自由基状态 由过氧化物酶反应产生，在溶液和晶体中。一个 辅助目标也是确定自由基损伤的性质 这导致了PGHS过氧化物酶的失活，并开发了一个模型 用于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.