Reduction-reoxidation cycles contribute to catalysis of disulfide isomerization by protein-disulfide isomerase

被引:113
|
作者
Schwaller, M [1 ]
Wilkinson, B [1 ]
Gilbert, HF [1 ]
机构
[1] Baylor Coll Med, Dept Biochem & Mol Biol, Verna & Marrs Mclean Dept Biochem, Houston, TX 77030 USA
关键词
D O I
10.1074/jbc.M211036200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:7154 / 7159
页数:6
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