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J. Biol. Chem., Vol. 278, Issue 9, 7154-7159, February 28, 2003
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From the Verna and Marrs McLean Department of Biochemistry and
Molecular Biology, Baylor College of Medicine,
Houston, Texas 77030
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.
Reduction-Reoxidation Cycles Contribute to Catalysis of disulfide
Isomerization by Protein-disulfide Isomerase*
,, and
*
This work was supported by National Institutes of Health
Grants R01-GM-40379 (to H. F. G. and M. S.) and T32-GM-08280 (to B. W.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Both authors contributed equally to this work.
§
To whom correspondence should be addressed: Dept. of Biochemistry
and Molecular Biology, Baylor College of Medicine, One Baylor Plaza,
Houston, TX 77030. Tel.: 713-798-5880; Fax: 713-796-9438; E-mail:hgilbert@bcm.tmc.edu.
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