Inhibition by dithiothreitol of the utilization of glutamine by carbamyl phosphate synthetase. Evidence for formation of hydrogen peroxide.

The glutaminase activity of glutamine-dependent carbamyl phosphate synthetase (Escherichia coli) and that of the separated light subunit of this enzyme, as well as the glutaminedependent synthetase activity catalyzed by the native enzyme, are inactivated by incubation in air with relatively low concentrations (0.025 to 1 mM) of dithiothreitol; oxidized dithiothreitol does not inhibit. Other mercaptans (glutathione, Z-mercaptoethanol, dithioerythritol) also inhibit. Low concentrations of dithiothreitol under nitrogen and high concentrations (5 to 25 mu) of dithiothreitol in air do not inactivate the glutamine-related functions of the separated light subunit or of the native enzyme. However, enzyme preparations that have been inhibited by low concentrations of dithiothreito1 in air are not readily reactivated by treatment with high concentrations of dithiothreitol. Incubation of the native enzyme with hydrogen peroxide (0.2 mM) also inhibits the glutamine-dependent activities. In distinction, treatment of the enzyme with either hydrogen peroxide or low concentrations of dithiothreitol in air does not aBect either the ammonia-dependent synthetase activity or the synthesis of ATP from ADP and carbamyl phosphate, while the bicarbonate-dependent hydrolysis of ATP is stimulated moderately by both of these reagents. The addition of catalase as well as the addition of L-albizziin, L-glutamine, EDTA, or a mixture of ATP, magnesium ions, bicarbonate, and L-ornithine protect against inhibition of the glutamine-dependent synthetase activity by dithiothreitol. Neither L-ornithine (a positive allosteric effector) nor UMP (a negative allosteric effector) significantly affects the inhibition while a high protein concentration (10 mg per ml) protects against inhibition. The glutaminase activity of the separated light subunit, like that of the intact enzyme, is inhibited by the glutamine analog L-2-amino-4-oxo-5-chloropentanoic acid (chloroketone); L-glutamine or L-albizziin can protect against this inhibition. [i4C]Chloroketone binds to the dithiothreitol-inhibited enzyme to the same extent as it does to the native enzyme; such binding is reduced in the presence of glutamine. The most straightforward interpretation of the present results is that metal ion-catalyzed oxidation of