Structural Insights into the Catalytic Mechanism of Human Glutamine Synthetase

NICOLAS CAMPOLO; RAFAEL RADI; FEDERICO M. ISSOGLIO; TILMAN GRUNE; SILVINA BARTESAGHI; ARI ZEIDA; DARIO ESTRIN

Tucumán

Congreso; IX Iberoamerican Congress of Biophysics; III Latin American Federation of Biophysical Societies (LAFeBS); XLV Annual Meeting of the Argentinian Biophysical Society (SAB); 2016

Glutamine synthetase (GS) catalyzes the formation of glutamine from glutamate and ammonia, using ATP as a cofactor. Glutamate is the major excitatory neurotransmitter, and in human's brain GS activity prevents glutamate dependent excitotoxicity (as also detoxifying ammonia). Neurodegenerative disorders such as Alzheimer's disease have been associated with the lack of GS activity. Moreover, GS activity has been shown to be affected by oxidative modifications, including tyrosine nitration in specific residues.The human enzyme has 373 residues per subunit and exists a decamer, formed by two stacked pentamers, with the active sites located at the interface of two adjacent monomers of the pentamer. The crystallographic structure was determined for several organisms, and mainly from this results a reaction mechanism is proposed. The overall reaction proceeds through a two step mechanism, involving first the ATP γ-phosphate transfer to the lateral chain of glutamate, followed by the nucleophilic attack of ammonia yielding glutamine and inorganic phosphate.In this work we present a detailed description of the reaction mechanism of human GS (HsGS) at an atomic level, as well as an exhaustive examination of structural properties closely related with the catalysis, using to this purpose molecular dynamics simulations (MD), and combined quantum mechanics and molecular mechanics simulations (QM/MM). Our results suggest that the first step in which the γ-glutamyl phosphate intermediate forms, has a 5 kcal/mol free energy barrier and a −8 kcal/mol reaction free energy, and then the second and rate-limiting step has a free energy barrier of 19 kcal/mol and a reaction free energy of almost zero. Also, these results were essential to achieve atomistic detailed information about how HsGS catalytic mechanism is affected by tyrosine nitration.