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+的尿素生化周期中，该酶 精氨酸酶通过通过 一种在分子水平上未知的机制。 这个反应 还提供l- or-氨酸氨酸作为多胺的生物合成前体， 精子和精子，重要的生长因子。 也是这样 反应的L-精氨酸的细胞内浓度受到影响，因此 介导一氧化氮的精氨酸依赖性形成。 一氧化氮 是大脑中重要的神经递质，是一种有效的细胞毒性剂 巨噬细胞，血管扩张剂和阴茎勃起的关键介体 哺乳动物。 肝脏中的精氨酸酶缺乏会导致高症血症，一种 遗传性疾病导致神经系统障碍和智力低下。 最近有几个因素聚集在一起，这为新的希望提供了 了解精氨酸酶水解活性的化学机制。 另外，细菌催化酶的前所未有的氧化还原活性。 这 提案描述了Dimanganese的结构和机械研究 精氨酸酶的活性位点。 与Temple的David Ash博士合作 大学我们建议使用蛋白质残基的位置定向诱变 与EPR和电子核双共振（Endor）结合使用 光谱镜：1）确定蛋白质残基 催化位点的两个锰离子，并分开精氨酸酶 来自锰催化酶，2）产品，尿素和l- or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 过氧化氢酶的活性及其与过氧化物酶活性的潜在联系和 无前体的合成，5）通过 锰配体的诱变，6）检查潜在精氨酸酶 新的Dimanganese综合体系列的活动 精氨酸酶的活性位点的良好结构模型。