The structure and function of a complex metalloflavoprotein

哺乳动物的酶催化次黄嘌呤和黄嘌呤的羟化反应，这是形成尿酸盐的最后两步，以脱氢酶形式(XDH)合成，但可以通过巯基残基的氧化或蛋白质分解很容易转化为氧化酶形式(XO)。XDH型催化剂在FAD反应部位倾向于NAD+还原，而XO排除型催化剂以氧气为底物，导致超氧阴离子和过氧化氢的生成。如果采取了适当的预防措施，这种蛋白质也可以以XDH的形式被分离出来，然后可以转化为表现出氧化酶行为的形式。由于从牛奶中提取脂肪球状膜中的蛋白质的方法有了重大改进，我们能够在含有抑制剂水杨酸盐的复合体中生长出具有衍射性的XDH和XO形式的晶体。本文报道了二聚体牛乳黄嘌呤氧化还原酶的晶体结构，其XDH值为2.1Å，XO值为2.5Å。对两种分子结构的比较表明，在蛋白质水解性诱导XDH型到XO型转变过程中发生的主要变化。二聚体的总尺寸为155Åx90Åx70Å。单体可分为三个结构域。XDH到XO的转化可以通过Cys和Cys 992的修饰可逆地发生，也可以通过Lys 551之后的蛋白水解酶不可逆地发生。鉴于这两种形式的酶之间普遍具有很高的结构保守性，我们仍在研究有关表面修饰的信息如何传递到埋藏的辅因子结合位点的潜在解释，从而导致环的构象发生重大移动和该位点的静电势发生逆转。然而，我们相信，正在进行的对提高分辨率的衍射数据的分析将提供这个问题的答案。

The mammalian enzymes, which catalyze the hydroxylation of hypoxanthine and xanthine, the last two steps in the formation of urate, are synthesized as the dehydrogenase form (XDH) but can be readily converted to the oxidase form (XO) by oxidation ofsulfhydryl residues or by proteolysis. XDH shows a preference for NAD+ reduction at the FAD reaction site, while XO exclusivery uses dioxygen as its substrate leading to the formation of superoxide anion and hydrogen peroxide. Provided proper precautions are used, this protein can also be isolated in its XDH form, which can then be converted to the form that exhibits oxidase behavior. Due to major improvements in the purification of the protein from fat globular membranes from cow's milk, we were able to grow diffraction-quality crystals of both the XDH and XO forms in complex with the inhibitor salicylate. We present the crystal structures of the dimericbovine milk xanthine oxidoreductase in its XDH form at 2.1Å and in its XO from at 2.5Å resolution. Comparison of the two molecular structures identifies the major changes that occur during the proteolyically induced XDH to XO transformation.The overall dimensions of the dimer are 155Åx90Åx70Å. The monomer can be divided into three domains. The conversion of XDH to XO may occur either reversibly by modification of Cys 535 and Cys 992, or irreversibly by proteolytic cleavage after Lys 551. Given the generally high structural conservation between the two forms of the enzyme, we are still investigating potential explanations of how the information about the surface modifications is transmitted to the buried cofactor binding site resulting in the major shift in conformation of a loop and a reversal of the electrostatic potential of this site. We are confident, however, that the ongoing analysis of diffraction data of improved resolution will provide answers to this question.