Schwaller Melissa, Wilkinson Bonney, Gilbert Hiram F
Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
J Biol Chem. 2003 Feb 28;278(9):7154-9. doi: 10.1074/jbc.M211036200. Epub 2002 Dec 15.
Protein-disulfide isomerase (PDI) catalyzes the formation and isomerization of disulfides during oxidative protein folding. This process can be error-prone in its early stages, and any incorrect disulfides that form must be rearranged to their native configuration. When the second cysteine (CGHC) in the PDI active site is mutated to Ser, the isomerase activity drops by 7-8-fold, and a covalent intermediate with the substrate accumulates. This led to the proposal that the second active site cysteine provides an escape mechanism, preventing PDI from becoming trapped with substrates that isomerize slowly (Walker, K. W., and Gilbert, H. F. (1997) J. Biol. Chem. 272, 8845-8848). Escape also reduces the substrate, and if it is invoked frequently, disulfide isomerization will involve cycles of reduction and reoxidation in preference to intramolecular isomerization of the PDI-bound substrate. Using a gel-shift assay that adds a polyethylene glycol-conjugated maleimide of 5 kDa for each sulfhydryl group, we find that PDI reduction and oxidation are kinetically competent and essential for isomerization. Oxidants inhibit isomerization and oxidize PDI when a redox buffer is not present to maintain the PDI redox state. Reductants also inhibit isomerization as they deplete oxidized PDI. These rapid cycles of PDI oxidation and reduction suggest that PDI catalyzes isomerization by trial and error, reducing disulfides and oxidizing them in a different configuration. Disulfide reduction-reoxidation may set up critical folding intermediates for intramolecular isomerization, or it may serve as the only isomerization mechanism. In the absence of a redox buffer, these steady-state reduction-oxidation cycles can balance the redox state of PDI and support effective catalysis of disulfide isomerization.
蛋白质二硫键异构酶（PDI）在氧化蛋白质折叠过程中催化二硫键的形成和异构化。此过程在早期阶段可能容易出错，任何形成的不正确二硫键都必须重新排列成其天然构象。当PDI活性位点中的第二个半胱氨酸（CGHC）突变为丝氨酸时，异构酶活性下降7 - 8倍，并且与底物的共价中间体积累。这导致有人提出第二个活性位点半胱氨酸提供了一种逃逸机制，防止PDI被缓慢异构化的底物困住（沃克，K.W.，和吉尔伯特，H.F.（1997年）《生物化学杂志》272，8845 - 8848）。逃逸也会减少底物，如果频繁调用，二硫键异构化将优先涉及还原和再氧化循环，而不是PDI结合底物的分子内异构化。使用一种凝胶迁移分析方法，为每个巯基添加一个5 kDa的聚乙二醇共轭马来酰亚胺，我们发现PDI的还原和氧化在动力学上是可行的，并且对异构化至关重要。当不存在氧化还原缓冲剂来维持PDI氧化还原状态时，氧化剂会抑制异构化并氧化PDI。还原剂也会抑制异构化，因为它们会耗尽氧化型PDI。这些PDI氧化和还原的快速循环表明，PDI通过反复试验催化异构化，还原二硫键并以不同构象氧化它们。二硫键的还原 - 再氧化可能为分子内异构化建立关键的折叠中间体，或者它可能是唯一的异构化机制。在没有氧化还原缓冲剂的情况下，这些稳态还原 - 氧化循环可以平衡PDI的氧化还原状态，并支持二硫键异构化的有效催化。
