Relationship between protein structure and methionine oxidation in recombinant human alpha 1-antitrypsin.
alpha 1-Antitrypsin is a metastable and conformationally flexible protein that belongs to the serpin family of protease inhibitors. Although it is known that methionine oxidation in the protein's active site results in a loss of biological activity, there is little specific knowledge regarding the reactivity of each of the protein's methionine residues. In this study, we have used peptide mapping to study the oxidation kinetics of each of alpha 1-antitrypsin's methionines in alpha 1-AT((C232S)) as well as M351L and M358V mutants. These kinetic studies establish that Met1, Met226, Met242, Met351, and Met358 are reactive with hydrogen peroxide at neutral pH and that each reactive methionine is oxidized in a bimolecular, rather than coupled, mechanism. Analysis of Met226, Met351, and Met358 oxidation provides insights regarding the structure of alpha 1-antitrypsin's active site that allow us to relate conformation to experimentally observed reactivity. The relationship between solution pH and methionine oxidation was also examined to evaluate methionine reactivity under conditions that perturb the native structure. Methionine oxidation data show that at pH 5, global conformational changes occur that alter the oxidation susceptibility of each of alpha 1-antitrypsin's 10 methionine residues. Between pH 6 and 9, however, more localized conformational changes occur that affect primarily the reactivity of Met242. In sum, this work provides a detailed analysis of methionine oxidation in alpha 1-antitrypsin and offers new insights into the protein's solution structure.
Griffiths
Steven W
SW
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA. swgriffi@mit.edu
Cooney
Charles L
CL
D003545:Cysteine / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D019272:Leukocyte Elastase / Q000037:antagonists & inhibitors; D008715:Methionine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012694:Serine / Q000235:genetics; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000515:alpha 1-Antitrypsin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D000515:alpha 1-Antitrypsin; D012694:Serine; D008715:Methionine; D019272:Leukocyte Elastase; D003545:Cysteine