Walker K W, Lyles M M, Gilbert H F
Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030, USA.
Biochemistry. 1996 Feb 13;35(6):1972-80. doi: 10.1021/bi952157n.
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.
蛋白质二硫键异构酶（PDI）是内质网中一种含量非常丰富的蛋白质，它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）《生物化学杂志》269，30946 - 30952]。每个二硫醇/二硫键活性位点都包含硫氧还蛋白共有序列CXXC。构建了仅含有单个活性位点半胱氨酸的蛋白质二硫键异构酶的四个突变体。对这些突变体的动力学分析表明，任一活性位点中的第一个（更靠近N端）半胱氨酸对于还原型牛胰核糖核酸酶A（RNase）重折叠过程中的氧化和重排催化至关重要。具有SGHC序列的突变活性位点即使在25微摩尔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（更靠近C端）半胱氨酸对于RNase二硫键重排的催化不是必需的，但对于RNase氧化的催化是必需的，即使在存在谷胱甘肽氧化还原缓冲液的情况下也是如此。具有CGHS序列的突变活性位点在RNase重折叠的重排阶段显示出野生型活性位点kcat活性的12% - 50%，但在氧化阶段活性<5%。此外，具有CGHS序列的突变体在稳态周转期间积累了大量的共价PDI - RNase复合物，而野生型酶和具有SGHC序列的突变体则没有。由于两个活性位点半胱氨酸对于二硫键形成的催化都是必需的，RNase氧化的主要机制可能涉及活性位点PDI二硫键的直接氧化。虽然它对于RNase重排的催化不是必需的，但更靠近C端的半胱氨酸确实对重排活性有2 - 8倍的贡献。提出了一种底物重排机制，其中第二个活性位点半胱氨酸为PDI提供了一种从不能及时重排的共价中间体“逃脱”的方式。第二个活性位点半胱氨酸通常可能作为野生型酶的内部时钟，限制分子内底物重排允许的时间。
