STRUCTURE/FUNCTION OF ARGINASE-EPR & ENDOR SPECTROSCOPY

精氨酸酶-EPR的结构/功能

• 批准号: 2144647
• 负责人: G CHARLES DISMUKES
• 金额: $ 11.69万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-02-01 至 1997-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2144647
• 关键词: active sites; arginase; catalase; chemical binding; electron nuclear double resonance spectroscopy; electron spin resonance spectroscopy; enzyme activity; enzyme mechanism; enzyme structure; hydrolysis; manganese; nitric oxide; peroxidases; protein structure function; site directed mutagenesis; stereochemistry

In the urea biochemical cycle for removal of toxic NG4+, the enzyme arginase catalyzes the hydrolysis of L-arginine to urea and L-ornithine by a mechanism which is not understood at the molecular level. This reaction also provides L-ornithine as biosynthetic precursor to the polyamines, spermine and spermidine, important growth factors. Also through this reaction the intracellular concentration of L-arginine is influenced, thus mediating the arginine-dependent formation of nitric oxide. Nitric oxide is an important neurotransmitter in the brain, a potent cytotoxic agent in macrophages, a vasodilator and the key mediator of penile erection in mammals. Arginase deficiency in the liver leads to hyperammonemia, an inherited disorder leading to neurologic impairment and mental retardation. Several factors have come together recently which offer new hope for understanding the chemical mechanism for arginase's hydrolytic activity. Additionally, an unprecedented redox activity of bacterial catalases. This proposal describes structural and mechanistic studies of the dimanganese active site of arginase. In collaboration with Dr. David Ash from Temple University we propose to use site-directed mutagenesis of protein residues in combination with EPR and Electron Nuclear Double Resonance (ENDOR) spectroscopies to: 1) identify the protein residues which coordinate the two manganese ions at the catalytic site and serve to distinguish arginase from manganese catalases, 2) the products, urea and L-ornithine, 3) determine the functions of both manganese ions in the catalysis of arginine hydrolysis, 4) characterize the chemical mechanism of the newly discovered catalase activity and its potential link to peroxidase activity and synthesis of NO precursors, 5) enhance the catalase activity of arginase by mutagenesis of the manganese ligands, 6) examine the potential arginase activity of a new family of dimanganese complexes which are known to be good structural models for the active site of arginase.

在去除有毒 NG4+ 的尿素生化循环中，酶 精氨酸酶催化L-精氨酸水解为尿素和L-鸟氨酸 一种在分子水平上尚未被理解的机制。 这个反应 还提供 L-鸟氨酸作为多胺的生物合成前体， 精胺和亚精胺，重要的生长因子。 也通过这个 反应影响细胞内L-精氨酸的浓度，因此 介导精氨酸依赖性一氧化氮的形成。 一氧化氮 是大脑中重要的神经递质，是一种有效的细胞毒剂 巨噬细胞，血管扩张剂和阴茎勃起的关键介质 哺乳动物。 肝脏精氨酸酶缺乏会导致高氨血症， 导致神经功能障碍和智力障碍的遗传性疾病。 最近有几个因素结合在一起，给我们带来了新的希望 了解精氨酸酶水解活性的化学机制。 此外，细菌过氧化氢酶具有前所未有的氧化还原活性。 这 提案描述了二锰的结构和机制研究 精氨酸酶的活性位点。 与天普大学的 David Ash 博士合作 大学我们建议使用蛋白质残基的定点诱变 与 EPR 和电子核双共振 (ENDOR) 相结合 光谱学：1）识别协调的蛋白质残基 催化位点上有两个锰离子，用于区分精氨酸酶 来自锰过氧化氢酶，2) 产品，尿素和 L-鸟氨酸，3) 确定两种锰离子在精氨酸催化中的功能 水解，4）表征新发现的化学机制 过氧化氢酶活性及其与过氧化物酶活性的潜在联系 NO前体的合成，5)增强精氨酸酶的过氧化氢酶活性 锰配体的诱变，6) 检查潜在的精氨酸酶 已知二锰络合物新家族的活性 精氨酸酶活性位点的良好结构模型。