ACTIVE SITES OF FREE RADICAL METALLOENZYMES

自由基金属酶的活性位点

• 批准号: 2184236
• 负责人: JAMES W WHITTAKER
• 金额: $ 16.53万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-02-01 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2184236
• 关键词: active sites; catalyst; chemical kinetics; chemical models; circular dichroism; electrochemistry; electron spin resonance spectroscopy; enzyme mechanism; enzyme substrate complex; free radical oxygen; galactose oxidase; intermolecular interaction; ligands; metalloenzyme; protein structure function; ribonucleotide reductase; spectrometry

Free radical biochemistry is an important and emerging new area probing the involvement of organic free radicals, molecules having unpaired electrons, in biochemical processes. Historically, studies on free radicals in biology have emphasized the deleterious effects of radicals in toxicity mechanisms and radiation damage, but there is a growing recognition of the essential role that free radicals play in enzyme catalysis and the biological function of redox metalloproteins. Early studies in the area of free radical enzymology focussed on Fe- and B12- dependent ribonucleotide reductases and mutases from certain anaerobic bacteria. However, the significance of radicals in biochemical mechanisms is now well established in enzymes with as important and diverse functions as prostaglandin and penicillin biosynthesis, lipid peroxidation and alkane oxidation. A motif which is emerging from these studies is the association of the radical site with a metal center that is involved in generation, stabilization or control of reactivity of radicals in proteins. Two of the most well-defined free radical metalloenzymes are galactose oxidase and the Fe-dependent ribonucleotide reductase RRB2. Each of these enzymes stabilize a protein side chain as a free radical in the resting enzyme, a radical which is required for catalytic activity. Extensive biochemical and basic spectroscopic characterization is now complete for both proteins, and both are also crystallographically defined at near atomic resolution. Detailed spectroscopic studies of these proteins can now be expected to extend to geometric information available in the crystalligraphic data to develop the special electronic structural features of the radical sites and metal interactions, leading to insights into the mechanism of stabilization of radicals in proteins and the control of their unique reactivity. these key aspects of free radical biochemistry have not been previously defined by protein structural studies. These key aspects of free radical biochemistry have not been previously defined by protein structural studies. Our studies will focus on the information contained in electron paramagnetic resonance (EPR), electronic absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of the active site complexes in galactose oxidase (GO), ribonucleotide reductase (RRB2) and isopenicillin N synthase (IPNS). In the former (GO, RRB2) we will probe the radical sites as well as the biological metal complex. For IPNS the resting ferrous enzyme and its substrate complex will be of key interest in terms of radical-generating species. In all three studies, inorganic models will provide important calibration. To probe the mechanisms of reactivity for free radical metalloenzymes we will explore chemical perturbations, examining the spectroscopic and electrochemical consequences of coordinating exogenous ligands in the active complexes, and investigate the details of turnover reaction using transient kinetics methods. These fundamental studies will form the basis for characterization of other important and interesting free radical metalloenzymes.

自由基生物化学是一个重要的、新兴的探索新领域 有机自由基的参与，具有不配对的分子 生化过程中的电子。从历史上看，关于自由的研究 生物学中的自由基强调了自由基的有害影响 毒性机制和辐射损伤，但越来越多 认识自由基在酶中发挥的重要作用 氧化还原金属蛋白的催化作用和生物学功能。早期的 自由基酶学领域的研究集中于 Fe- 和 B12- 来自某些厌氧菌的依赖性核糖核苷酸还原酶和变位酶 细菌。然而，自由基在生化中的重要性 现在，酶的机制已得到很好的确立，具有同样重要和重要的作用 前列腺素和青霉素生物合成、脂质等多种功能 过氧化和烷烃氧化。从这些中出现的主题 研究是自由基位点与金属中心的关联 参与反应性的产生、稳定或控制 蛋白质中的自由基。两种最明确的自由基 金属酶是半乳糖氧化酶和铁依赖性核糖核苷酸 还原酶RRB2。这些酶中的每一种都稳定蛋白质侧链： 静息酶中的自由基，这是维持酶活性所需的自由基 催化活性。广泛的生化和基础光谱 两种蛋白质的表征现已完成，并且两种蛋白质也都已完成 以近原子分辨率进行晶体学定义。详细的 这些蛋白质的光谱研究现在有望扩展到 晶体数据中可利用的几何信息可用于开发 自由基位点的特殊电子结构特征和 金属相互作用，从而深入了解金属相互作用的机制 蛋白质中自由基的稳定及其独特的控制 反应性。自由基生物化学的这些关键方面尚未被研究 先前由蛋白质结构研究定义。这些关键方面 自由基生物化学之前尚未由蛋白质定义 结构研究。我们的研究将集中于所包含的信息 在电子顺磁共振（EPR）、电子吸收、 圆二色性（CD）和磁圆二色性（MCD）光谱 半乳糖氧化酶 (GO)、核糖核苷酸中的活性位点复合物 还原酶 (RRB2) 和异青霉素 N 合酶 (IPNS)。在前者中（GO， RRB2）我们将探测自由基位点以及生物金属 复杂的。对于 IPNS，静息亚铁酶及其底物复合物 就产生自由基的物种而言，将引起人们的主要兴趣。总共 三项研究中，无机模型将提供重要的校准。到 探究自由基金属酶的反应机制 将探索化学扰动，检查光谱和 协调外源配体的电化学后果 活性复合物，并使用研究周转反应的细节 瞬态动力学方法。这些基础研究将形成 其他重要且有趣的免费表征的基础 自由基金属酶。