Oxidative inactivation of glutamine synthetase from the cyanobacterium Anabaena variabilis.
In crude extracts of the cyanobacterium Anabaena variabilis, glutamine synthetase (GS) could be effectively inactivated by the addition of NADH. GS inactivation was completed within 30 min. Both the inactivated GS and the active enzyme were isolated. No difference between the two enzyme forms was seen in sodium dodecyl sulfate-gels, and only minor differences were detectable by UV spectra, which excludes modification by a nucleotide. Mass spectrometry revealed that the molecular masses of active and inactive GS are equal. While the Km values of the substrates were unchanged, the Vmax values of the inactive GS were lower, reflecting the inactivation factor in the crude extract. This result indicates that the active site was affected. From the crude extract, a fraction mediating GS inactivation could be enriched by ammonium sulfate precipitation and gel filtration. GS inactivation by this fraction required the presence of NAD(P)H, Fe3+, and oxygen. In the absence of the GS-inactivating fraction, GS could be inactivated by Fe2+ and H2O2. The GS-inactivating fraction produced Fe2+ and H2O2, using NADPH, Fe3+, and oxygen. Accordingly, the inactivating fraction was inhibited by catalase and EDTA. This GS-inactivating system of Anabaena is similar to that described for oxidative GS inactivation in Escherichia coli. We conclude that GS inactivation by NAD(P)H is caused by irreversible oxidative damage and is not due to a regulatory mechanism of nitrogen assimilation.
Martin
G
G
Lehrstuhl für Physiologie und Biochemie der Pflanzen, Universität Konstanz, Germany.
Haehnel
W
W
Böger
P
P
D017033:Anabaena / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005974:Glutamate-Ammonia Ligase / Q000037:antagonists & inhibitors*; D007700:Kinetics; D009243:NAD / Q000494:pharmacology; D009249:NADP / Q000494:pharmacology; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism
D004791:Enzyme Inhibitors; D009243:NAD; D009249:NADP; D010088:Oxidoreductases; D005974:Glutamate-Ammonia Ligase