Physiochemical Studies on Xanthine Oxidizing Enzymes

黄嘌呤氧化酶的理化研究

• 批准号: 9313781
• 负责人: Vincent Massey
• 金额: $ 30万
• 依托单位: Regents of the University of Michigan - Ann Arbor
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 1998-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9313781&HistoricalAwards=false
• 关键词: Physiochemical; Studies; Xanthine; Oxidizing; Enzymes

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. ***

9313781 Massey 我们已成功分离出稳定状态的牛奶黄嘌呤脱氢酶，该酶可以可逆地转化为氧化酶形式，其中首选电子受体是 02，而不是脱氢酶形式的 NAD。 迄今为止，我们的工作表明，这种可逆相互转化的主要影响是由脱氢酶向氧化酶转化中空间邻近硫醇残基的氧化引起的，涉及蛋白质的构象变化，这主要影响黄素氧化还原中心。 然而，快速反应研究表明钼蝶呤氧化还原中心也可能受到相互转化的影响。 该提案的主要目的是详细研究两种形式酶的理化性质，目的是定义赋予两种形式不同催化性质的参数。 这项研究将广泛使用快速反应分光光度法、EPR 和 EXAFS 测量、黄素替代研究、酶与不同类别底物的催化特性，以及酶的有效抑制剂的结合模式的检查。 %%% 黄嘌呤氧化酶通常被认为与氧化应激现象有关，其中氧自由基与活体组织的许多重要成分发生反应，包括蛋白质、脂质和 DNA。 这种氧化损伤与癌症、缺血和衰老过程有关。 黄嘌呤氧化酶以两种形式存在，氧化酶以将氧还原为超氧自由基 (02-) 为代价来氧化黄嘌呤和多种嘌呤和嘧啶，而脱氢酶则氧化相同的化合物，但使用 NAD（维生素 B3 的辅酶衍生物）代替氧。 我们从牛奶中分离出这种酶，并表明这两种形式可以以可逆的方式相互转化，具体取决于蛋白质硫醇残基的氧化还原状态。 这两种酶形式具有许多不同的特性，这显然是由于不同的蛋白质构象所致。 虽然过去对氧化酶形式进行了广泛的研究，但对脱氢酶的了解相对较少。 本研究旨在详细探讨这两种形式的性质差异，以便为有关其生理功能的概念提供坚实的科学基础。 ***