Many enzymes are now known to be subject to a covalent modification mediated by mixed function oxidation. This modification may have physiologic and pathologic significance in diverse processes. These include the regulation of protein turnover, accumulation of modified proteins during aging, killing of pathogens by host defense mechanisms, limitation of autolysis, pulmonary damage by smoking and air pollutants, and in oxygen toxicity. Multiple oxidative modifications may be introduced into a protein. Glutamine synthetase was subjected to varying times of exposure to mixed function oxidation to provide samples of graded oxidation. Amino acid analysis revealed loss of two histidine residues. No other changes in amino acid composition were detected. The enzyme lost both catalytic activity and a divalent metal binding site upon oxidation of the first histidine residue. This form of the enzyme was not susceptible to proteolytic degradation by several purified proteases. Oxidation of the second histidine residue rendered the enzyme susceptible to degradation. Studies of the surface hydrophobicity of glutamine synthetase revealed that oxidative modification modulates that hydrophobicity. Initial oxidation converts the protein to a more hydrophilic species which is not a substrate for a purified protease. Additional oxidation generates a more hydrophobic form which is a substrate. Studies with a transition-state analog demonstrated that occupancy of the active site blocks oxidative modification and prevents the changes normally induced by oxidation. Occupancy of the active site provides a mechanism by which cellular metabolites may regulate mixed function oxidation of specific proteins.