Clarifying the Catalytic Mechanism of Human Glutamine Synthetase: A QM/MM Study

Glutamine synthetase (GS) is a crucial enzyme responsible for the elimination of both neurotoxic glutamate and toxic ammonium, by combining them into glutamine. Alterations on the GS activity are associated with severe liver and neurodegenerative diseases and its absence or malformation results in death. In this work, the catalytic mechanism of human GS has been investigated with high-level QM/MM calculations, showing a two-phase reaction cycle. During phase 1, GS activates the reactants (NH₄⁺ and glutamate) with extreme efficiency, through NH₄⁺ deprotonation by E305 and glutamate phosphorylation by ATP, in two spontaneous and barrierless reactions. At phase 2, NH₃ attacks the γ-glutamyl phosphate being concomitantly deprotonated by the leaving PO₄^3-, forming the glutamine and HPO₄^2- products. The second phase contains the rate limiting step, with a ΔG^‡ of 19.2 kcal·mol^-1 associated with the nucleophilic substitution of the phosphate by NH₃. The final reaction free energy is -34.5 kcal·mol^-1. Both phases are exergonic, the first by -22.9 kcal·mol^-1 and the second by -11.6 kcal·mol^-1. Direct NH₄⁺ attack is shown to be inefficient; the possible bases that perform the NH₄⁺ deprotonation were systematically investigated. Negative E305 was shown to be the only one possibly responsible for NH₄⁺ deprotonation. Altogether, these results provide a clear atomic level picture of the reaction cycle of GS, consistent with experimental and theoretical studies on GS of this and other organisms, and provide the necessary insights for the development of more specific therapeutic GS inhibitors.