A SPECTROELECTROCHEMICAL STUDY OF SELECTED FLAVOPROTEINS

黄蛋白催化许多重要的生物氧化还原反应。 了解反应机理是最重要的。因为 氧化还原电势(E‘)是对 酶，它是重要的，并将作为进一步的参考 学习。先前的一项研究表明，蛋白质基质调节着 黄素的氧化还原性质和催化活性。为了支持…… 这个想法包括氧化还原数据、动力学数据和结构数据 检测出黄毒素和葡萄糖氧化酶。总而言之，这些数据具有 提供了对黄素蛋白机制的重要见解。这些研究 强调有必要非常详细地研究氧化还原电位 黄素蛋白。 尽管氧化还原性质E‘和n(数目 电子转移)，没有对黄素蛋白氧化还原进行系统研究 之前已经制作了一些属性。我们开发了一种新的 光谱电化学法研究化合物的氧化还原性质 黄素蛋白。通过使用这种方法，我们能够获得第一个 黄素蛋白氧化酶E‘和n的测定。氧化还原数据 与最新可用的结构数据很好地关联在这些 酵素。然而，很难将氧化还原数据与 已知的这些酶的催化机制。我们现在建议一种方法，用这种方法 这两者可能是有关联的。我们认为底物与酶的结合 进一步“调节”酶的能量水平。如果这个假设是 正确的，底物结合酶和抑制剂结合酶的E‘和n值 应该不同于我们的游离酶的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’)来检验我们的假设 N)氧化酶和其他黄素蛋白类结合的成员 底物和抑制剂。我们将继续系统地衡量 游离黄素蛋白的氧化还原特性。底物的酶调节 细胞色素P450cam的结合已被确定。这项规定是 由自由和底物结合的氧化还原电位测量确定 酵素。我们自己的工作表明，竞争性抑制剂的结合 D-氨基酸氧化酶显著改变了E‘和n。因此，我们 解决有关酶调节的基本问题。电化学法 方法是为这些问题提供答案的最佳方法。

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.