The thioester formation mechanism proposed in this work (Figure ) begins from the enzyme-substrate (ES) complex with deprotonation of Cys84 by His178 to form the thiolate in Int1. The nucleophilic Cys84 attacks the substrate carbonyl forming a tetrahedral oxyanion intermediate (Int2). The His178 imidazolium transfers a proton to the substrate to form an ammonium in (Int3). Lastly, the tetrahedral The electronic energy profiles of Active and Inactive models (Figure ) illustrate how differences in active site geometry substantially influence the reaction. Deprotonation of Cys84 in Active has a barrier of 14.5 kcal/mol, significantly higher than the 3.2 kcal/mol required for Inactive. This difference is due to the positioning of the Gln substrate. In the optimized Active-ES, the substrate is observed in a near-attack conformation, with the carbonyl carbon 3.56 Å from the Cys84 sulfur and the NH2 group hydrogen bonding with the His178 Nε (2.01 Å). This interaction is disrupted in the Active-TS1 when the His178 Nε accepts the proton from Cys84. Alternatively, in the Inactive-ES the substrate is positioned farther away (3.84 Å), which enables Cys84 to hydrogen bond with His178 (1.91 Å). The Gln pose optimized in Inactive is not possible in Active because the proximity of fGln123 confines the substrate. The rate of thioester formation in the Active geometry is determined by the collapse of the tetrahedral oxyanion intermediate, with a TS bond breaking distance of 2.08 Å (Active-TS4, Figure ). This yields a computationally predicted barrier of ∆E ‡ =18.7 kcal/mol, which agrees well with the experimental rate of T. maritima (0.67 ± 0.02 s -1 at 298.15 K, corresponding to a barrier of around 17.7 kcal/mol). Isotope effects of the amide nitrogen on glutamine bound to a similar class-I GAT, carbamyl phosphate synthetase (CPS), support the release of ammonia as the rate-limiting step. Thioester formation was also found to be rate-limiting in cysteine esterase. In the Inactive geometry, the formation of ammonium (Inactive-TS3, Figure ) is highest in energy, yielding a barrier of ∆E ‡ =30.5 kcal/mol for thioester formation. This barrier is larger than experimental measurements (21.1 and 22.7 kcal/mol). Alternative mechanistic pathways were explored, such as concerted NH3 formation and tetrahedral collapse, but were not located. Another possibility is that more energetically favorable structural rearrangements occur along the reaction coordinate to enable catalysis. In support of this, the barrier for the Val51 backbone flip in the absence of PrFAR is estimated by steered MD to be approximately 22 kcal/mol, which aligns well with the experimental rate. Therefore, our current DFT results suggest that the enzymatic reaction rate in the absence of the allosteric effector is limited by the barrier for conformational interconversion rather than by the elementary bond-forming and breaking steps in the active site. The elementary step most affected by an active site structural change is the formation of the tetrahedral acyl-enzyme via TS2. This transformation requires 13.6 kcal/mol from Int1 in Active and nearly twice as much in Inactive (26.1 kcal/mol). This step demonstrates the importance of IGPS active site geometry in stabilizing the incipient oxyanion.