Physiochemical Studies on Xanthine Oxidizing Enzymes

9313781 Massey我们已经成功地分离了稳定状态的牛奶黄嘌呤脱氢酶，它可以可逆地转化为氧化酶形式，其中优选的电子受体是O2，而不是NAD，因为它是脱氢酶形式。 迄今为止，我们的工作表明，这种可逆的相互转化，这是由氧化的空间相邻的巯基残基的脱氢酶氧化酶转换所带来的主要影响，涉及蛋白质的构象变化，主要影响黄素氧化还原中心。 然而，有迹象表明，从快速反应研究表明，该dopterin氧化还原中心也可能受到相互转换。 该提案的主要目的是详细研究两种形式的酶的物理化学性质，目的是定义赋予两种形式如此不同的催化性质的参数。 这项研究将涉及广泛使用的快速反应分光光度法，EPR和EXAFS测量，黄素替代研究，催化性质的酶与不同类别的基板，并检查的模式结合的酶的有效抑制剂。 黄嘌呤氧化酶通常被认为与称为氧化应激的现象有关，其中氧的自由基物质与活组织的许多基本成分反应，包括蛋白质，脂质和DNA。 这种氧化损伤与癌症、局部缺血和衰老过程有关。 黄嘌呤氧化酶以两种形式存在：氧化酶，其以将氧还原成超氧自由基（O2-）为代价来氧化黄嘌呤和各种嘌呤和嘧啶;以及脱氢酶，其氧化相同的化合物，但使用NAD（维生素B3的辅酶衍生物）代替氧。 我们已经从牛奶中分离出了这种酶，并表明这两种形式可以以可逆的方式相互转化，这取决于蛋白质巯基残基的氧化还原状态。 这两种酶形式具有许多不同的性质，这显然是由于不同的蛋白质构象。 虽然氧化酶形式在过去已经被广泛研究，但相对而言，对脱氢酶知之甚少。 本研究旨在详细探讨这两种形态在性质上的差异，为其生理功能的概念提供坚实的科学依据。 ***

9313781 Massey We have succeeded in isolating milk xanthine dehydrogenase in a stable state, which can be converted reversibly into the oxidase form, where the preferred electron acceptor is 02, rather than NAD, as it is with the dehydrogenase form. Our work to date indicates that the major effects of this reversible interconversion, which is brought about by oxidation of spatially vicinal thiol residues in the dehydrogenase to oxidase conversion, involve conformational changes in the protein which affect mainly the flavin redox center. However, there are indications from rapid reaction studies that the molybdopterin redox center may also be affected by the interconversion. The major thrust of this proposal 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 will involve extensive use of rapid reaction spectrophotometry, EPR and EXAFS measurements, flavin replacement studies, catalytic properties of the enzymes with different classes of substrates, and examination of the modes of binding of potent inhibitors of the enzymes. %%% The enzyme xanthine oxidase is commonly believed to be implicated in the phenomenon known as oxidative stress, in which free radical species of oxygen react with many essential components of living tissues, including proteins, lipids and DNA. Such oxidative damage has been implicated in cancer, ischemia and the aging process. Xanthine oxidase exists in two forms, the oxidase, which oxidizes xanthine and a wide variety of purines and pyrimidines at the expense of reducing oxygen to the superoxide radical (02-), and the dehydrogenase, which oxidizes the same compounds, but uses NAD, a coenzyme derivative of vitamin B3, instead of oxygen. We have isolated the enzyme from cow's milk, and shown that the two forms can be interconverted in a reversible manner, depending on the oxidation-reduction state of the protein thiol residues. The two enzyme forms have many different properties, which are clearly due to different protein conformations. While the oxidase form has been studied extensively in the past, comparatively little is known of the dehydrogenase. This study is to explore in detail the differences in properties of the two forms, in order to provide a firm scientific basis for concepts regarding their physiological functions. ***