Oxidation of the active site of glutamine synthetase: Conversion of arginine-344 to γ-glutamyl semialdehyde

Abstract

Metal-catalyzed oxidative modification of proteins is implicated in a number of physiologic and pathologic processes. The reaction is presumed to proceed via a site-specific free radical mechanism, with the site-specificity conferred by a cation-binding site on the protein. The oxidation of bacterial glutamine synthetase has been studied in detail, providing the opportunity to examine whether the oxidation is consistent with a site-specific radical reaction. Oxidation leads to the appearance of carbonyl groups in amino acid side chains of the protein, and labeling of those carbonyl groups with fluoresceinamine facilitated purification of the oxidized peptide from a tryptic digest. The oxidized residue was arginine-344, which was converted to a γ-glutamyl semialdehyde residue. Histidine-269 had previously been shown to be converted to asparagine during metal-catalyzed oxidation. Both arginine-344 and histidine-269 are situated at the metal-nucleotide binding pocket of the enzyme's active site, thus establishing the site-specificity of the oxidation.