BIOCHEMISTRY AND HEPATIC TOXICITY OF CARBON RADICALS

描述（由申请人提供）：自由基过程与越来越多的病理学有关，包括主要的健康问题，如癌症和动脉粥样硬化。然而，尽管在过去的几年里取得了令人印象深刻的进展，蛋白质自由基的基本生物化学仍然没有完全理解。本申请中提出的工作依赖于在支持到期期间取得的关键进展，包括（a）鉴定乳过氧化物酶中血红素基团的自催化共价结合中的残基，（B）鉴定乳过氧化物酶中在酶与H2 O2反应时转化为自由基中心的特定残基，（c）将辣根过氧化物酶突变成在其共价结合其血红素的能力方面模拟哺乳动物过氧化物酶的酶，以及（d）在高铁肌红蛋白系统中证明酪氨酸氧化成自由基取决于与氧化中心的距离以外的因素。本项目集中在蛋白质自由基生物化学的两个方面：（a）羧酸根，一个高度被忽视的物种，在过氧化物酶功能的可能形成和作用，和（B）形成不寻常的分子内交联及其功能后果的机制。本项目的第一个目的是通过辣根过氧化物酶模型系统的研究来确定羧酸基团在血红素-蛋白质交联反应中的作用。第二个目的是使用相同的模型来澄清自由基在髓过氧化物酶的甲硫氨酸-乙烯基键形成中的作用。第三个目的是澄清为什么共价血红素结合是重要的哺乳动物过氧化物酶功能。第四个目的是研究活性位点羧酸基团的作用和过氧化氢酶-过氧化物酶中形成不寻常交联的机制。最终的目标是采用附加酚的高铁肌红蛋白模型系统来探索确定蛋白质中哪些酪氨酸被氧化的关系。这些研究应阐明在生理和病理过程中过氧化产生的碳自由基的产生和命运，并为调节或抑制这些过程的方法提供见解。

DESCRIPTION (provided by applicant): Free radical processes have been implicated in a growing range of pathologies, including major health problems such as cancer and atherosclerosis. Nevertheless, despite impressive progress in the past few years, the basic biochemistry of protein radicals continues to be incompletely understood. The work proposed in this application rests on key advances made in the expiring period of support, including (a) identification of the residues in autocatalytic covalent binding of the heme group in lactoperoxidase, (b) Identification of specific residues in lactoperoxidase that are converted to free radicals centers upon reaction of the enzyme with H202, (c) mutation of horseradish peroxidase into an enzyme that mimics the mammalian peroxidases in its ability to bind its heme covalently, and (d) demonstration in a metmyoglobin system that the oxidation of tyrosines to radicals depends on factors other than distance from the oxidizing center. The present project focuses on two aspects of protein radical biochemistry: (a) the possible formation and role of carboxylate radicals, a highly neglected species, in peroxidase function, and (b) the mechanism of formation of unusual intramolecular cross-links and their functional consequences. The first aim of the present project is to define the role of the carboxylate group in the heme-protein cross-linking reaction through studies of the horseradish peroxidase model system. The second aim is to use the same model to clarify the role of radicals in formation of the methionine-vinyl bond of myeloperoxidase. The third aim is to clarify why covalent heme binding is important for mammalian peroxidase function. The fourth aim is to examine the role of the active site carboxylate group and the mechanism(s) by which the unusual cross-links in catalase-peroxidase enzymes are formed. A final goal is to employ a metmyoglobin model system with appended phenols to explore the relationships which determine which tyrosines are oxidized in a protein. These studies should shed light on the generation and fates of peroxidatively generated carbon radicals in physiological and pathological processes and provide insights into approaches for the modulation or suppression of such processes.