The reduction potential of the active site disulfides of human protein disulfide isomerase limits oxidation of the enzyme by Ero1

Chambers, J.E., Tavender, T.J., Oka, O.B.V. , Warwood, S., Knight, D. and Bulleid, N.J. (2010) The reduction potential of the active site disulfides of human protein disulfide isomerase limits oxidation of the enzyme by Ero1. Journal of Biological Chemistry, 285(38), pp. 29200-29207. (doi: 10.1074/jbc.M110.156596)

Abstract

Disulfide formation in newly synthesized proteins entering the mammalian endoplasmic reticulum is catalyzed by protein disulfide isomerase (PDI), which is itself thought to be directly oxidized by Ero1α. The activity of Ero1α is tightly regulated by the formation of noncatalytic disulfides, which need to be broken to activate the enzyme. Here, we have developed a novel PDI oxidation assay, which is able to simultaneously determine the redox status of the individual active sites of PDI. We have used this assay to confirm that when PDI is incubated with Ero1α, only one of the active sites of PDI becomes directly oxidized with a slow turnover rate. In contrast, a deregulated mutant of Ero1α was able to oxidize both PDI active sites at an equivalent rate to the wild type enzyme. When the active sites of PDI were mutated to decrease their reduction potential, both were now oxidized by wild type Ero1α with a 12-fold increase in activity. These results demonstrate that the specificity of Ero1α toward the active sites of PDI requires the presence of the regulatory disulfides. In addition, the rate of PDI oxidation is limited by the reduction potential of the PDI active site disulfide. The inability of Ero1α to oxidize PDI efficiently likely reflects the requirement for PDI to act as both an oxidase and an isomerase during the formation of native disulfides in proteins entering the secretory pathway.

• Item Type: Articles
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Bulleid, Professor Neil and Tavender, Dr Timothy and Oka, Dr Ojore
• Authors: Chambers, J.E., Tavender, T.J., Oka, O.B.V., Warwood, S., Knight, D., and Bulleid, N.J.
• Subjects: Q Science > QH Natural history > QH345 Biochemistry
• College/School: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Journal Name: Journal of Biological Chemistry
• Journal Abbr.: J Biol Chem.
• Publisher: American Society for Biochemistry and Molecular Biology, Inc.
• ISSN: 0021-9258
• ISSN (Online): 1083-351X