Physiochemical Studies on Xanthine Oxidizing Enzymes

9313781梅西我们已经成功地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物型，其中首选的电子受体是02，而不是脱氢酶型的NAD。到目前为止，我们的工作表明，这种可逆的相互转换的主要影响是脱氢酶到氧化酶转化中空间邻近的硫醇残基的氧化，涉及蛋白质的构象变化，主要影响黄素氧化还原中心。然而，来自快速反应研究的迹象表明，钼掺杂蛋白的氧化还原中心也可能受到相互转化的影响。这项建议的主要目的是详细研究这两种形式的酶的物理化学性质，目的是确定使这两种形式具有如此不同催化性质的参数。这项研究将涉及快速反应分光光度法的广泛应用，EPR和EXAFS测量，黄素替代研究，酶与不同类型底物的催化性能，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，在氧化应激中，氧的自由基物种与生命组织的许多基本成分发生反应，包括蛋白质、脂类和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，一种是将黄嘌呤和多种嘌呤和嘧啶氧化成超氧自由基(02-)，而另一种是脱氢酶，它氧化相同的化合物，但使用的是NAD，即维生素B3的辅酶衍生物，而不是氧。我们已经从牛奶中分离出这种酶，并证明这两种形式可以以可逆的方式相互转化，这取决于蛋白质硫醇残基的氧化还原状态。这两种酶有许多不同的性质，这显然是由于不同的蛋白质构象所致。虽然过去人们对脱氢酶的形态进行了广泛的研究，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态在性质上的差异，为认识它们的生理功能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. ***