BIOCHEMISTRY AND HEPATIC TOXICITY OF CARBON RADICALS

碳自由基的生物化学仍然知之甚少，尽管 碳自由基作为机械探针的独特价值及其直接意义 与氧自由基病理学的相关性。这里提出的工作是建立在 在支助期满期间取得的以下主要进展：(A) 证实了一个事实，即自由基几乎总是 由过氧化物酶产生，但很少由单加氧酶产生，(B)成功 具有催化活性的辣根过氧化物酶(HRP)和 杆状病毒系统中的乳过氧化物酶，以及(C)初步探索 不同部位辣根过氧化物酶的构效关系 特定的突变作用。拟议工作的第一个目标是进一步 重点阐述原型过氧化物酶hrp的功能 关于(A)支配催化剂周转的结构特征和 底物专一性，(B)底物自由基的机制 (C)单加氧酶反应的催化作用。第二 我们的目标是扩大我们对植物和真菌的了解 生理学上的两种过氧化物酶：乳过氧化物酶和髓过氧化物酶 重要的哺乳动物酶，重点是(A)性质和机制 人工血红素基团与蛋白质的共价结合，(B) 两个氧化等价物在被氧化的2电子中的位置 中间体，称为化合物I，以及(C)使 卤化物离子的氧化。第三个目标是探索 过氧化生成的自由基的生物命运，特别是它们的 与蛋白质的反应。这些研究的集体意图是 促进我们对形成自由基物种的酶和 这些自由基与蛋白质的反应。这一结果应该有助于 阐明过氧化生成的自由基在毒理学中的作用 并揭示抑制这类进程的机制。

The biochemistry of carbon radicals remains poorly understood despite the unique value of carbon radicals as mechanistic probes and their immediate relevance to oxygen radical pathology. The work proposed here builds on the following key advances made during the expiring period of support: (a) validation of a rationale for the fact that radicals are almost invariably produced by peroxidases but rarely by monooxygenases, (b) successful expression of catalytically active horseradish peroxidase (HRP) and lactoperoxidase in a baculovirus system, and (c) initial exploration of the structure-activity relationships for horseradish peroxidase by site specific mutagenesis. The first goal of the proposed work is to further elucidate the function of HRP, the prototypical peroxidase, with emphasis on (a) the structural features that govern catalytic turnover and substrate specificity, (b) the mechanism of substrate free radical generation, and (c) the catalysis of monooxygenase reactions. The second goal is to extend our growing understanding of plant and fungal peroxidases to lactoperoxidase and myeloperoxidase, two physiologically important mammalian enzymes, with emphasis on (a) the nature and mechanism of covalent binding of the prosthetic heme group to the protein, (b) the location of the two oxidation equivalents in the 2-electron oxidized intermediate known as compound I, and (c) the features that make possible the oxidation of halide ions. The third goal is to explore aspects of the biological fates of peroxidatively generated radicals, particularly their reactions with proteins. The collective intent of these studies is to advance our understanding of the enzymes that form radical species and of the reactions of these radicals with proteins. The results should help to clarify tic role of peroxidatively generated radicals in toxicological processes and to uncover mechanisms for the suppression of such processes.