Catalysis of oxidative protein folding by mutants of protein disulfide isomerase with a single active-site cysteine.
Catalysis of oxidative protein folding by mutants of protein disulfide isomerase with a single active-site cysteine.
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DOI：
10.1021/bi952157n
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发表时间：
1996-02
期刊：
影响因子：
2.9
通讯作者：
K. Walker;M. Lyles;H. Gilbert
中科院分区：
文献类型：
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作者：
K. Walker;M. Lyles;H. Gilbert
蛋白质二硫键异构酶（PDI）是内质网中非常丰富的蛋白质，其利用位于两个不同硫氧还蛋白同源结构域中的两个不等价的氧化还原活性位点促进二硫键的形成和重排[莱尔斯，M. M.，&吉尔伯特，H. F.（1994）J.Biol.Chem.269，30946-30952]。每个二硫醇/二硫化物活性位点含有硫氧还蛋白共有序列CXXC。构建了四种蛋白质二硫键异构酶突变体，它们只有一个活性位点半胱氨酸。这些突变体的动力学分析表明，第一个（更多的N-末端）半胱氨酸在任何一个活性位点是必不可少的还原牛胰腺核糖核酸酶A（RNase）的重折叠过程中的氧化和重排的催化。具有序列SGHC的突变体活性位点即使在25 μ M的浓度下也未显示出可检测到的二硫键形成或重排活性。第二个（更C-末端）半胱氨酸对于RNA酶二硫键重排的催化不是必需的，但它对于RNA酶氧化的催化是必需的，即使在谷胱甘肽氧化还原缓冲液的存在下。具有序列CGHS的突变活性位点在RNase重折叠的重排阶段显示野生型活性位点的12%-50%的kcat活性，但在氧化阶段显示< 5%的活性。此外，具有序列CGHS的突变体在稳态周转期间积累显著水平的共价PDI-RNA酶复合物，而野生型酶和具有序列SGHC的突变体则不。由于两个活性位点半胱氨酸对于催化二硫化物形成是必不可少的，所以RNA酶氧化的主要机制可能涉及通过活性位点PDI二硫化物的直接氧化。虽然它不是催化RNA酶重排所必需的，但C端半胱氨酸越多，对重排活性的贡献就越大。底物重排的机制，其中第二个活性位点的半胱氨酸提供了一种方式来“逃避”的共价中间体，不及时重排PDI的建议。第二个活性位点半胱氨酸通常可以作为野生型酶的内部时钟，限制分子内底物重排的时间。
Protein disulfide isomerase (PDI), a very abundant protein in the endoplasmic reticulum, facilitates the formation and rearrangement of disulfide bonds using two nonequivalent redox active-sites, located in two different thioredoxin homology domains [Lyles, M. M., & Gilbert, H. F. (1994) J. Biol. Chem. 269, 30946-30952]. Each dithiol/disulfide active-site contains the thioredoxin consensus sequence CXXC. Four mutants of protein disulfide isomerase were constructed that have only a single active-site cysteine. Kinetic analysis of these mutants show that the first (more N-terminal) cysteine in either active site is essential for catalysis of oxidation and rearrangement during the refolding of reduced bovine pancreatic ribonuclease A (RNase). Mutant active sites with the sequence SGHC show no detectable activity for disulfide formation or rearrangement, even at concentrations of 25 microM. The second (more C-terminal) cysteine is not essential for catalysis of RNase disulfide rearrangements, but it is essential for catalysis of RNase oxidation, even in the presence of a glutathione redox buffer. Mutant active sites with the sequence CGHS show 12%-50% of the kcat activity of wild-type active sites during the rearrangement phase of RNase refolding but < 5% activity during the oxidation phase. In addition, mutants with the sequence CGHS accumulate significant levels of a covalent PDI-RNase complex during steady-state turnover while the wild-type enzyme and mutants with the sequence SGHC do not. Since both active-site cysteines are essential for catalysis of disulfide formation, the dominant mechanism for RNase oxidation may involve direct oxidation by the active-site PDI disulfide. Although it is not essential for catalysis of RNase rearrangements, the more C-terminal cysteine does contribute 2-8-fold to the rearrangement activity. A mechanism for substrate rearrangement is suggested in which the second active-site cysteine provides PDI with a way to "escape" from covalent intermediates that do not rearrange in a timely fashion. The second active-site cysteine may normally serve the wild-type enzyme as an internal clock that limits the time allowed for intramolecular substrate rearrangements.