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.

在尿素生物化学循环中去除有毒的NG 4+， 精氨酸酶催化L-精氨酸水解为尿素和L-鸟氨酸， 这是一种在分子水平上尚不清楚的机制。 该反应 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 L-精氨酸的细胞内浓度受到影响，因此 介导一氧化氮的烟碱依赖性形成。 一氧化氮 是大脑中一种重要的神经递质，是一种强效的细胞毒性剂， 巨噬细胞，血管扩张剂和阴茎勃起的关键介质， 哺乳动物 肝脏精氨酸酶缺乏导致高氨血症， 导致神经损伤和智力迟钝的遗传性疾病。 最近有几个因素结合在一起，为...... 了解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 该提案描述了二锰的结构和机理研究 酶的活性位点。 与坦普尔的大卫阿什博士合作 我们建议使用蛋白质残基的定点突变 结合EPR和电子核双共振（ENDOR） 1）鉴定与蛋白质配位的蛋白质残基， 两个锰离子在催化位点，并用于区分脱氢酶 来自锰过氧化氢酶，2）产物，尿素和L-鸟氨酸，3） 测定了两种锰离子在精氨酸催化反应中的作用 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）通过抑制NO前体的合成来增强过氧化氢酶的活性。 锰配体的诱变，6）检查潜在的磷酸酶 活性的一个新的家庭的二锰配合物，这是已知的是 为酶活性中心提供了良好的结构模型