We partially purified a preparation from Escherichia coli that proteolytically degrades the enzyme glutamine synthetase [L-glutamate:ammonia' ligase (ADP-forming), EC 6.3.1.2]. The degradation is at least a two-step process. First, the glutamine synthetase undergoes an oxidative modification. This modification leads to loss of catalytic activity and also renders the protein susceptible to proteolytic attack in the second step. The oxidative step displays characteristics of a mixed-function oxidation, requiring both molecular oxygen and a reduced nucleotide. This step can also be catalyzed by a purified, mammalian cytochrome P-450 system, as well as by a model system consisting of ascorbic acid and oxygen. Catalase blocks this oxidative modification step. Thus, the overall process of proteolytic degradation can be observed only if care is taken to remove catalase activity from the extracts. The inactivation reaction is dependent on the state of adenylylation of the glutamine synthetase, suggesting that this is a physiologically important reaction. If so, then mixed-function oxidases are now implicated in the process of intracellular protein turnover.The biochemical pathways for degradation of cellular proteins remain uncharted. Regulation of these pathways will likely emerge as an important mechanism of the control of cellular metabolism. Glutamine is a pivotal compound in nitrogen metabolism (1). Not surprisingly, glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] from Gramnegative bacteria is subject to exquisite regulation, including derepression/repression, feedback inhibition, and covalent modification. This modification (adenylylation) is in turn intricately controlled by a cascade mechanism (1). Because the concentration of a given enzyme is determined by the rates of its synthesis and degration (2), it seemed likely that the level of glutamine synthetase in these bacteria would be regulated by an enzymically mediated degradative mechanism.We show here that glutamine synthetase is degraded in Klebsiella aerogenes and by a partially purified preparation from Escherichia coli. This degradation involves two steps. First, glutamine synthetase undergoes an oxidative inactivation. Second, proteolysis occurs. The inactivation step displays characteristics of a mixed-function oxidation and can be catalyzed by a purified mammalian cytochrome P-450 system. A model system consisting simply of ascorbic acid and 02 can also carry out the oxidative inactivation. After inactivation 'by the P450 or ascorbate.systems, glutamine synthetase is then susceptible to proteolytic degradation by bacterial extracts.

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