STRUCTURAL BIOLOGY OF PEROXIDATION BY PGH SYNTHASES

PGH 合成酶过氧化的结构生物学

• 批准号: 6107867
• 负责人: R Michael Garavito
• 金额: $ 10.47万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-06-01 至 2000-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6107867
• 关键词: Raman spectrometry; X ray crystallography; active sites; chemical binding; chemical transfer reaction; computer simulation; drug interactions; electron spin resonance spectroscopy; enzyme activity; free radicals; heme; human tissue; isozymes; membrane proteins; nonsteroidal antiinflammatory agent; oxidative stress; peroxidation; prostaglandin endoperoxide synthase; protein binding; protein structure function; site directed mutagenesis; structural biology; ultraviolet spectrometry

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.

我们持续暴露于氧自由基和相关氧化剂可以启动 与衰老有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 -2是整合膜血红素酶，可转化花生四烯酸 进入Prostaglandin Cascade的前体。 PGHS同工型 产生自由基，损坏细胞和细胞成分并激活 环境污染物。 PGHS过氧化物酶构成一个独特的和 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，而且还与 大型哺乳动物过氧化物酶。第二，对 酶的结构是理解机制的先决条件 产生氧化产物。 酶 - 配体相互作用和结构/功能的性质 天然PGHS过氧化物酶以及现场定向的关系 将使用停止流量紫外线吸收来探索突变体 光谱，共振拉曼光谱和X射线晶体学。这 具体目标如下。首先，我们将调查 酶 - 配体相互作用和相关的结构功能关系 在PGHS同工型的天然过氧化物酶中以表征血红素铁 通过使用血红素铁配体的环境，然后识别然后 表征使用弱（即 更稳定的）还原剂，例如2-氨基氟和苄基羟 酸。其次，我们将研究中的结构/功能关系 卵子PGHS-1过氧化物酶通过过氧化物酶部位的突变分析。 第三，我们将研究人类的结构功能关系 PGHS-2过氧化物酶通过检查具有等效突变的修饰PGHS-2 如Ovine PGHS-1。最后，我们将尝试遵循结构 自由基生成涉及的变化以阐明 自由基转移和过氧化物酶依赖性灭活。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，溶液和晶体中产生。一个 子公司的目标也是确定自由基损害的性质 这导致PGHS过氧化物酶失活并开发模型 在其他生物系统中蛋白质的自由基损害。