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 reductionoxidation cycles can balance the redox state of PDI and support effective catalysis of disulfide isomerization.The folding of proteins destined for the secretory pathway occurs in the endoplasmic reticulum where a quality control system ensures that secreted proteins are correctly folded (1), including the correct formation of disulfide bonds. Disulfides that form early in the folding process are often incorrect; cysteines can be mispaired (2), or disulfides can be formed in the wrong temporal order, making it difficult to oxidize buried cysteines (3). To rectify these errors, the incorrect disulfides must be broken and new ones formed in a different configuration.The endoplasmic reticulum contains folding assistants that help proteins achieve their correct disulfide arrangement. Protein-disulfide isomerase (PDI) 1 is a 55-kDa protein of the endoplasmic reticulum which catalyzes disulfide formation (oxidase activity) as well as the rearrangement of incorrect disulfide pairings (isomerase activity) (4), accelerating both processes without drastically altering the refolding pathway (1). PDI catalyzes the chemical changes but does not appear to guide the process or unfold misfolded substrates actively (5, 6). PDI has two active sites, one near the amino terminus and the other near the carboxyl terminus (7). Each active site contains two cysteines in...
Sacchromyces cerevisiae protein disulfide isomerase (yPDI) was expressed in the E. coli periplasm by using plasmids encoding the OmpA-yPDI-(His)(6) fusion gene under the control of the araBAD, trc, or T7 promoter. The expression levels of yeast PDI under these promoters were compared. Our results showed that yeast PDI expressed into the periplasm could catalyze the formation of disulfide bonds in alkaline phosphatase, restoring the phoA(+) phenotype in dsbA(-) mutants. The yeast PDI was purified from the Escherichia coli periplasm and shown to exhibit catalytic properties comparable to those of the rat enzyme with reduced RNase as substrate. In vivo, coexpression of the yeast PDI increased the yield of bovine pancreatic trypsin inhibitor (BPTI) in E. coli by 2-fold, similar to the effect seen previously with the coexpression of the rat enzyme. However yeast PDI was more effective than rat PDI in facilitating the expression of active tissue plasminogen activator (tPA). These results point to differences in the substrate specificity of various PDI enzymes, at least in the context of the E. coli periplasm.
Protein disulfide isomerase is a multifunctional protein that facilitates the formation of disulfide crosslinks between cysteine residues during the early stages of protein folding and secretion. It is a member of a large family of oxidoreductases that catalyse exchange reactions between thiols and disulfides.
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