Rasmussen J T, Rasmussen M S, Petersen T E
Protein Chemistry Laboratory, University of Aarhus, Denmark.
J Dairy Sci. 2000 Mar;83(3):499-506. doi: 10.3168/jds.S0022-0302(00)74909-5.
Mammalian xanthine oxidoreductase exists intracellularly in its dehydrogenase form. However, outside of this reducing milieu the enzyme quickly transforms into an oxidase form. Interconversion can be controlled by sulfhydryl reactive reagents, suggesting that disulfide bridging is linked to this phenomenon. The present work identified cysteines involved in the interconversion process. Purified enzyme was subjected to mild reduction with 1,4-dithioerythriol to regain dehydrogenase activity, and the accessible cysteines were labeled with specific radioactive alkylation reagents, iodoacetic acid. This partial alkylation stabilizes the dehydrogenase form, presumable by hindering formation of disulfide bond(s). Six of 38 cysteines were found to be labeled (residues 169, 170, 535, 992, 1317, and 1325). The significance of this labeling of bovine xanthine oxidoreductase is discussed in relation to structural knowledge about the enzyme, and especially by comparison with the AA sequences of avian and invertebrate enzymes, which do not undergo conversion. Cysteines 535 and 992 are the most likely marked residues to be involved in the interconversion, whereas the other cysteines are located too far from the cofactorbinding areas in xanthine oxidoreductase.
哺乳动物的黄嘌呤氧化还原酶在细胞内以脱氢酶形式存在。然而，在这种还原环境之外，该酶会迅速转变为氧化酶形式。相互转化可由巯基反应性试剂控制，这表明二硫键桥连与这一现象有关。目前的研究确定了参与相互转化过程的半胱氨酸。用1,4 - 二硫代赤藓糖醇对纯化的酶进行轻度还原以恢复脱氢酶活性，并用特异性放射性烷基化试剂碘乙酸标记可及的半胱氨酸。这种部分烷基化可能通过阻碍二硫键的形成来稳定脱氢酶形式。在38个半胱氨酸中发现有6个被标记（残基169、170、535、992、1317和1325）。结合该酶的结构知识，特别是与不发生转化的鸟类和无脊椎动物酶的氨基酸序列进行比较，讨论了牛黄嘌呤氧化还原酶这种标记的意义。半胱氨酸535和992最有可能是参与相互转化的标记残基，而其他半胱氨酸距离黄嘌呤氧化还原酶中的辅因子结合区域太远。
