2 - Oxidative Inactivation of Human Glutamine Synthetase: Biochemical and Computational Studies

Abstract

Glutamine synthetase (GS) is a key metabolic enzyme that catalyzes the ATP-dependent synthesis of glutamine from glutamate and ammonia. In the central nervous system, it is mainly located in the cytosol of astrocytes, playing an important role in ammonia detoxification and prevention of glutamate-dependent excitotoxicity. Alterations in GS activity may lead to astroglial dysfunction, affecting neuronal function and survival. In its native state, GS has a decameric structure, with a monomer molecular weight of 42 KDa. Several in vitro and in vivo studies in plants, bacteria and mammals, have shown that GS activity is highly susceptible to biologically-relevant reactive oxygen species, in particular peroxynitrite (ONOO–). Peroxynitrite-derived radicals promote tyrosine nitration yielding 3-nitrotyrosine (3-NT). Tyrosine nitration of GS has been identified as one of the main oxidative modifications associated to enzyme inactivation in pathological conditions; however, the critical residues involved and the molecular mechanisms participating in GS inactivation are still undefined. Herein we have worked with human glutamine synthetase (hGS), which was expressed in E. coli and further purified to homogeneity. Then, we studied the effect of different oxidants on hGS function and structure, paying special attention to the oxidative mechanisms of inactivation, by combining classical biochemical assays with molecular dynamic simulations. Peroxynitrite addition caused a dose-dependent inactivation of hGS, associated to an increase in 3-NT levels. In addition, we were able to detect the formation of high molecular weight aggregates, resistant to the action of reductants, probably due to di-tyrosine crosslinks. Both, tyrosine nitration and aggregate-formation strongly correlated with enzyme inactivation, however, pH-dependent studies suggested that di-tyrosine formation had a larger impact on enzyme activity, compared to nitration. Under these conditions we also observed thiol-oxidation and disulfide formation, although these modification did not impact on enzyme activity. In addition, peptide-mapping MS analysis identified critical modified residues, namely Tyr 185 and 269. In parallel to the experimental studies, molecular dynamic simulations were performed to understand the catalytic mechanisms of the reaction with the goal of defining, with an atomic level of detail, how the oxidative postranslational modifications affect activity, in particular tyrosine residues associated to the substrate binding sites.