HEME ENZYME ENGINEERING

血红素酶工程

• 批准号: 2444727
• 负责人: THOMAS L POULOS
• 金额: $ 21.77万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-07-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2444727
• 关键词: Escherichia coli; L ascorbate oxidase; X ray crystallography; aminoacid; cytochrome c; cytochrome oxidase; electron transport; enzyme mechanism; heme; mutant; protein engineering; protein structure function; site directed mutagenesis

DESCRIPTION: Heme proteins are ideally suited for detailed structure-function studies using protein engineering techniques. Most well studied heme proteins (globins, c-and b-type cytochromes, peroxidases, P450) all have exactly the same heme prosthetic group yet each exhibits very different and very clearly defined functions. These differences are dictated by the interaction between the protein and heme much of which has been deciphered through a variety of spectral probes and x-ray crystallography. Therefore, there are some specific questions that can be asked regarding the relationship between structure and function in heme proteins. The research in this proposal will focus on peroxidase. Recombinant systems have been developed for two peroxidases: cytochrome c and ascorbate peroxidase. Ascorbate peroxidase is the first and, to date, only eukaryotic heme enzyme that has been expressed as a holo-enzyme in E. coli. One goal of this proposal is to fully characterize the ascorbate peroxidase and then use mutagenesis to study its function. This will complement the ongoing work on cytochrome c peroxidase where mutagenesis will be used to probe the mechanism of intraprotein electron transfer. Since the substrate specificities of both peroxidases are markedly different and we have the crystal structures of both, we are in a good position to employ mutagenesis methods to decipher what structural features control substrate specificity.

描述：血红素蛋白非常适合详细 使用蛋白质工程技术的结构功能研究。 最顺利 研究了血红素蛋白（Globins，C-and B型细胞色素，过氧化物酶，P450） 所有人都有完全相同的血红素假肢组 不同且非常明确的功能。 这些差异是 由蛋白质与血红素之间的相互作用决定的大部分 通过各种光谱探针和X射线解密 晶体学。 因此，有一些具体的问题 问有关血红素结构与功能之间的关系 蛋白质。 该提案中的研究将集中在过氧化物酶上。 已开发了针对两个过氧化酶的重组系统：细胞色素c 和抗坏血酸过氧化物酶。 抗坏血酸过氧化物酶是第一个，迄今为止 仅在E中表示为全酶的真核血红素酶 大肠杆菌。 该提案的目标之一是充分表征抗坏血酸 过氧化物酶，然后使用诱变研究其功能。 这会 补充诱变的细胞色素C过氧化物酶的正在进行的工作 将用于探测肾上蛋白电子转移的机理。 由于两个过氧化物酶的底物特异性都明显不同 而且我们具有两者的晶体结构，我们处于一个很好的位置 采用诱变方法破译哪些结构特征控制 底物特异性。