The Reactivity and Oxidation Pathway of Cysteine 232 in Recombinant Human α1-Antitrypsin
Oxidative damage to the sulfur-containing amino acids, methionine and cysteine, is a major concern in biotechnology and medicine. α1-Antitrypsin, which is a metastable and conformationally flexible protein that belongs to the serpin family of protease inhibitors, contains nine methionines and a sing...
Saved in:
| Main Author: | |
|---|---|
| Other Authors: | , |
| Format: | article |
| Published: |
2002
|
| Online Access: | https://hdl.handle.net/11073/25757 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| Summary: | Oxidative damage to the sulfur-containing amino acids, methionine and cysteine, is a major concern in biotechnology and medicine. α1-Antitrypsin, which is a metastable and conformationally flexible protein that belongs to the serpin family of protease inhibitors, contains nine methionines and a single cysteine in its primary sequence. Although it is known that methionine oxidation in the protein active site results in a loss of biological activity, there is little specific knowledge regarding the reactivity of its unpaired thiol, Cys-232. In this study, the thiol-modifying reagent NBD-Cl (7-chloro-4-nitrobenz-2-oxa-1,3-diazole) was used to label peroxide-modified α1-antitrypsin and demonstrate that the Cys-232in vitro oxidation pathway begins with a stable sulfenic acid intermediate and is followed by the formation of sulfinic and cysteic acid in successive steps. pH-dependent reactivity with hydrogen peroxide showed that Cys-232 has a pK a of 6.86 ± 0.05, a value that is more than 1.5 pH units lower than that of a typical protein thiol. pH-induced conformational changes in the region surrounding Cys-232 were also examined and indicate that mildly acidic conditions induce a conformation that enhances Cys-232 reactivity. In summary, this work provides new insights into α1-antitrypsin reactivity in oxidizing environments and shows that a unique structural environment renders its unpaired thiol, Cys-232, its most reactive amino acid. |
|---|