Physicochemical Studies of Xanthine Oxidizing Enzymes

电话：96-03591本研究分离出稳定状态下的牛奶黄嘌呤脱氢酶，它可以可逆地转化为氧化形式，其中首选的电子受体是氧而不是NAD，因为它是脱氢酶形式。这项工作表明，这种由脱氢酶中空间相邻的硫醇残基氧化到氧化酶转化的可逆相互转化的主要影响涉及蛋白质的构象变化，主要影响黄素氧化还原中心。这项研究的主要目的是详细研究两种形式的酶的物理化学性质，目的是确定赋予两种形式如此不同的催化性质的参数。这项研究包括广泛使用快速反应分光光度法，黄素替代研究，酶与不同类型底物的催化特性，以及酶的结合模式和有效抑制剂的机制的检查。2. 黄嘌呤氧化酶能以两种稳定的形式存在，并具有不同的催化性质，这使它成为一种非常不寻常的酶。人们普遍认为，这种相互转化形成了各种氧化应激现象的基础，因为氧化酶形式具有产生氧自由基的能力。为了评估这些假设，有必要对这两种酶形式的性质有一个彻底的了解。本项目就是针对这样的研究展开的。

96-03591 Massey 1. Technical This investigator has isolated milk xanthine dehydrogenase in a stable state, which can be converted reversibly into the oxidized form, where the preferred electron acceptor is oxygen rather than NAD, as it is with the dehydrogenase form. This work indicates that the major effects of this reversible interconversion, which is brought about by oxidation of spatially adjacent thiol residues in the dehydrogenase to oxidase conversion, involve conformational changes in the protein which affect mainly the flavin redox center. The major thrust of this research is to study in detail the physicochemical properties of the two forms of the enzyme, with the aim of defining the parameters that give the two forms such different catalytic properties. This study involves extensive use of rapid reaction spectrophotometry, flavin replacement studies, catalytic properties of the enzymes with different classes of substrates, and examination of the modes of binding and mechanism of potent inhibitors of the enzymes. 2. Nontechnical The ability of xanthine oxidase to exist in two stable forms with very different catalytic properties makes it a very unusual enzyme. It is widely believed that this interconversion forms the basis of a variety of oxidative stress phenomena, because of the ability of the oxidase form to generate oxygen radicals. In order to assess such hypotheses, it is essential to have a thorough understanding of the properties of the two enzyme forms. This project is focused on such a study.