Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom.
J Biol Chem. 2010 Sep 17;285(38):29200-7. doi: 10.1074/jbc.M110.156596. Epub 2010 Jul 23.
Disulfide formation in newly synthesized proteins entering the mammalian endoplasmic reticulum is catalyzed by protein disulfide isomerase (PDI), which is itself thought to be directly oxidized by Ero1α. The activity of Ero1α is tightly regulated by the formation of noncatalytic disulfides, which need to be broken to activate the enzyme. Here, we have developed a novel PDI oxidation assay, which is able to simultaneously determine the redox status of the individual active sites of PDI. We have used this assay to confirm that when PDI is incubated with Ero1α, only one of the active sites of PDI becomes directly oxidized with a slow turnover rate. In contrast, a deregulated mutant of Ero1α was able to oxidize both PDI active sites at an equivalent rate to the wild type enzyme. When the active sites of PDI were mutated to decrease their reduction potential, both were now oxidized by wild type Ero1α with a 12-fold increase in activity. These results demonstrate that the specificity of Ero1α toward the active sites of PDI requires the presence of the regulatory disulfides. In addition, the rate of PDI oxidation is limited by the reduction potential of the PDI active site disulfide. The inability of Ero1α to oxidize PDI efficiently likely reflects the requirement for PDI to act as both an oxidase and an isomerase during the formation of native disulfides in proteins entering the secretory pathway.
新合成的进入哺乳动物内质网的蛋白质中的二硫键形成由蛋白质二硫键异构酶（PDI）催化，而 PDl 本身被认为是直接被 Ero1α 氧化。Ero1α 的活性受到非催化性二硫键形成的严格调节，这些二硫键需要断裂以激活酶。在这里，我们开发了一种新的 PDI 氧化测定法，该方法能够同时确定 PDI 各个活性位点的氧化还原状态。我们使用该测定法证实，当 PDI 与 Ero1α 孵育时，只有 PDI 的一个活性位点被直接缓慢氧化。相比之下，Ero1α 的无调控突变体能够以与野生型酶相当的速率氧化 PDI 的两个活性位点。当 PDI 的活性位点突变为降低其还原电势时，野生型 Ero1α 现在可以以 12 倍的活性将两者都氧化。这些结果表明，Ero1α 对 PDI 活性位点的特异性需要存在调节性二硫键。此外，PDI 氧化的速率受到 PDI 活性位点二硫键还原电势的限制。Ero1α 不能有效地氧化 PDI 可能反映了 PDI 在进入分泌途径的蛋白质中原位二硫键形成过程中既充当氧化酶又充当异构酶的要求。
