OP-38 - Insights into the mechanisms of peroxynitrite-mediated inactivation of human glutamine synthetase

Abstract

Glutamine synthetase (GS) is a key metabolic enzyme that catalyzes the ATP-dependent synthesis of glutamine from glutamate and ammonia. In the CNS, it is mainly located in the cytosol of astrocytes, playing an important role in detoxification of ammonia and excess glutamate. Alterations in GS activity and oxidative posttranslational modifications have been found in vivo. Herein, we studied the effect of oxidants on recombinant hGS function and structure with a combination of experimental and computational studies. Peroxynitrite caused a dose-dependent inactivation of hGS and 3-nitrotyrosine (NT) formation. In addition, higher molecular weight aggregates were formed likely due to 3,3´-dityrosine crosslinks. pH studies suggested that 3,3´-dityrosine formation had a larger impact on GS activity than nitration. MS analysis identified some critical residues modified, namely Tyr 185 and 269. H 2 O 2 -dependent thiol oxidation did not impact on GS activity. Exposure of human astrocytes to SIN-1 led to GS nitration and decreased enzyme activity. Molecular dynamic studies in GS (Issoglio et al. Biochemistry 2016) are being performed in nitrated forms of the enzyme to define with an atomic level of detail how tyrosine oxidative modifications affect GS structure and activity.