The turnover of Escherichia coli glutamine synthetase occurs in two steps. First, the glutamine synthetase is oxidatively modified, rendering it enzymatically inactive; and second, the modifed glutamine synthetase is degraded. The oxidation step, catalyzed by several mixed function oxidase systems, results in the loss of a single histidine and the generation of a carbonyl group. We proposed that the oxidation step marks glutamine synthetase for degradation. The purpose of this project is to isolate and characterize the protease activity which degrade the modified glutamine synthetase. A protease that degrades the modified GS 10-20 times more rapidly than native has been purified from E. coli extracts. The protease precipitates in a 60-70% ammonium sulfate cut, does no bind to DEAE ion exchanger at pH 7.5, binds to an HPLC Phenyl column, and to an HPLC DEAE column at pH 9. The purified protease migrates on HPLC gele chromatography as a single protein peak with a molecular weight of 70,000-80,000. The protease activity is optimum at pH 9, and its isoelectric pH is 7-7.4. It is activated 2-4 fold by 50 MuM zinc plus 1 mM magnesium. The protease is inhibited by aprotinin but not by other serine protease inhibitors. Metal chelators and thiol reagents also inactivate the protease, and these effects are not reversible.