Oxidation of the active site of glutamine synthetase: conversion of arginine-344 to gamma-glutamyl semialdehyde.
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 fluorescein-amine facilitated purification of the oxidized peptide from a tryptic digest. The oxidized residue was arginine-344, which was converted to a gamma-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.
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
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D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism; D001419:Bacteria / Q000201:enzymology; D001665:Binding Sites; D005456:Fluorescent Dyes; D005974:Glutamate-Ammonia Ligase / Q000737:chemistry / Q000378:metabolism*; D005971:Glutamates / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry
D005456:Fluorescent Dyes; D005971:Glutamates; D010446:Peptide Fragments; C021199:glutamic acid gamma-semialdehyde; D006639:Histidine; D001120:Arginine; D005974:Glutamate-Ammonia Ligase