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Exploring the Catalytic Mechanism of Human Glutamine Synthetase by Computer Simulations
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posted on 2016-09-29, 00:00 authored by Federico
M. Issoglio, Nicolas Campolo, Ari Zeida, Tilman Grune, Rafael Radi, Dario A. Estrin, Silvina BartesaghiGlutamine
synthetase is an important enzyme that catalyzes the
ATP-dependent formation of glutamine from glutamate and ammonia. In
mammals, it plays a key role in preventing excitotoxicity in the brain
and detoxifying ammonia in the liver. In plants and bacteria, it is
fundamental for nitrogen metabolism, being critical for the survival
of the organism. In this work, we show how the use of classical molecular
dynamics simulations and multiscale quantum mechanics/molecular mechanics
simulations allowed us to examine the structural properties and dynamics
of human glutamine synthetase (HsGS), as well as
the reaction mechanisms involved in the catalytic process with atomic
level detail. Our results suggest that glutamine formation proceeds
through a two-step mechanism that includes a first step in which the
γ-glutamyl phosphate intermediate forms, with a 5 kcal/mol free
energy barrier and a −8 kcal/mol reaction free energy, and
then a second rate-limiting step involving the ammonia nucleophilic
attack, with a free energy barrier of 19 kcal/mol and a reaction free
energy of almost zero. A detailed analysis of structural features
within each step exposed the relevance of the acid–base equilibrium
related to protein residues and substrates in the thermodynamics and
kinetics of the reactions. These results provide a comprehensive study
of HsGS dynamics and establish the groundwork for
further analysis regarding changes in HsGS activity,
as occur in natural variants and post-translational modifications.