The reactivity and oxidation pathway of cysteine 232 in recombinant human alpha 1-antitrypsin.
Oxidative damage to the sulfur-containing amino acids, methionine and cysteine, is a major concern in biotechnology and medicine. alpha1-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 alpha1-antitrypsin and demonstrate that the Cys-232 in 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 alpha1-antitrypsin reactivity in oxidizing environments and shows that a unique structural environment renders its unpaired thiol, Cys-232, its most reactive amino acid.
Griffiths
Steven W
SW
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge 02139, USA. swgriffi@mit.edu
King
Jonathan
J
Cooney
Charles L
CL
D003545:Cysteine / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000515:alpha 1-Antitrypsin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004220:Disulfides; D011994:Recombinant Proteins; D000515:alpha 1-Antitrypsin; D003545:Cysteine