A SPECTROELECTROCHEMICAL STUDY OF SELECTED FLAVOPROTEINS

选定黄素蛋白的光谱电化学研究

• 批准号: 3276903
• 负责人: MARIAN T. STANKOVICH
• 金额: $ 3.8万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-07-01 至 1988-01-19
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3276903
• 关键词: NAD(P)H dehydrogenase; acidity /alkalinity; aminoacid oxidase; bacteria; electrical potential; electrochemistry; electron transport; enzyme structure; enzyme substrate; flavoproteins; hydroxybenzoate; oxidation reduction reaction; spectrometry; thermodynamics

Flavoproteins catalyze many important biological redox reactions. Understanding the mechanism of reaction is of primary importance. Because the redox potential (E') is a measure of the equilibrium energy level of the enzyme, it is important and will serve as a reference for further studies. A previous study indicates the protein matrix regulates both the redox properties and the catalytic activities of the flavin. In support of this idea the redox data, kinetic data, and structural data have been obtained for flavodoxin and glucose oxidase. Taken together this data has provided important insights into flavoprotein mechanisms. These studies stress the need for a very detailed study of redox potentials of the flavoproteins. Despite the importance of the redox properties E' and n (number of electrons transferred), no systematic study of flavoprotein redox properties has previously been made. We developed a new spectroelectrochemical method for studying the redox properties of flavoproteins. By using this method we were able to obtain the first measurements of E' and n for the flavoprotein oxidases. The redox data correlates very well with newly available structural data on these enzymes. However, it is difficult to correlate the redox data with the known catalytic mechanism for these enzymes. We now suggest a way in which the two can be related. We propose that binding of substrate to the enzyme further "tunes" the energy levels of the enzymes. If this hypothesis is correct, the E' and n values of substrate and inhibitor bound enzymes should be different from the E' and n values of the free enzymes which we have already measured. We propose to test our hypothesis by measuring the redox properties (E' and n) of both oxidases and members of the other flavoprotein classes bound to substrates and inhibitors. We will continue our systematic measurement of the free flavoprotein redox properties. Enzyme regulation by substrate binding has been determined for cytochrome P450 cam. That regulation was identified by redox potential measurements of free and substrate bound enzyme. Our own work has shown that the binding of competitive inhibitors to D-amino acid oxidase significantly alters both E' and n. Thus, we are addressing basic questions about enzyme regulation. The electrochemical method is the best method of providing answers to these questions.

黄素蛋白催化许多重要的生物氧化还原反应。 了解反应机理是至关重要的。 因为 氧化还原电位（E '）是平衡能级的量度， 酶，它是重要的，并将作为进一步的参考 问题研究 先前的研究表明，蛋白质基质调节着细胞的 氧化还原性质和催化活性。 支持 这个想法的氧化还原数据，动力学数据，和结构数据已经 获得黄素氧还蛋白和葡萄糖氧化酶。 综合这些数据， 对黄素蛋白的机制提供了重要的见解。 这些研究 强调需要非常详细的研究氧化还原电位的 黄素蛋白 尽管氧化还原性质E'和n（氧化还原的数量）是重要的， 电子转移），没有系统的研究黄素蛋白氧化还原 以前有过财产。 我们开发了一种新的 光谱电化学方法研究的氧化还原性能 黄蛋白。 通过使用这种方法，我们能够获得第一个 黄素蛋白氧化酶的E'和n的测量。 氧化还原数据 与这些新的结构数据非常吻合 内切酶 然而，很难将氧化还原数据与氧化还原数据相关联。 这些酶的已知催化机制。 我们现在提出一种方法， 这两者是有联系的。 我们认为底物与酶的结合 进一步“调谐”酶的能量水平。 如果这个假设是 正确，底物和抑制剂结合酶的E'和n值 应该不同于我们所用的游离酶的E'和n值， 已经测量过了。 我们建议通过测量氧化还原性质（E'和E'）来测试我们的假设。 n）氧化酶和其他黄素蛋白类的成员， 底物和抑制剂。 我们将继续系统地测量 游离黄素蛋白的氧化还原性质。 底物对酶的调节 已经确定了细胞色素P450 cam的结合。 这一规定是 通过游离和底物结合的氧化还原电位测量确定 酵素 我们自己的研究表明竞争性抑制剂的结合 对D-氨基酸氧化酶的反应显著改变E'和n。 因此，我们 解决酶调节的基本问题。 电化学 方法是回答这些问题的最佳方法。