Detailed Study of Human Glutamine Synthetase by QM/MM Simulations

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

Rio de Janeiro

Congreso; V Latin American Protein Society Meeting; 2016

Glutamine synthetase (GS) is an essential enzyme in nitrogen metabolism that catalyzes the ATP-dependent synthesis of glutamine from glutamate and ammonia. Given the high toxicity of ammonia to animals, GS acts as an important pathway for ammonia detoxification. In the brain it has also a key function at sustaining the glutamate-glutamine cycle avoiding glutamate excitotoxicity. In the present work, we show how the use of multiple steered molecular dynamics simulations (MSMD) and combined quantum mechanics with molecular mechanics scheme (QM/MM), allowed us to examine human GS (HsGS) reaction mechanisms involved in the catalytic process with atomic level detail. Glutamine formation proceeds through a two step mechanism which includes first the γ-glutamyl phosphate intermediate formation, with a 5 kcal/mol energy barrier and -8 kcal/mol reaction free energy, and then the ammonia nucleophilic attack over the intermediate being the rate limiting step, with an energy barrier of 19 kcal/mol and a reaction free energy of zero. Also, supported with classical molecular dynamics (MD) simulations results, a detailed analysis of structural features within each step exposed the impact of different protonation states related to protein residues and substrates over the reaction thermodynamics and kinetics.