Imidazole glycerol phosphate synthase (IGPS) is a glutamine amidotransferase (GAT) vital to purine and histidine biosynthetic pathways in bacteria, fungi, and plants, making it an attractive antimicrobial therapeutic target. IGPS from Thermotoga maritima is a heterodimer composed of the HisH and HisF subunits (HisFH) (Figure ). HisH performs glutamine (L-Gln) hydrolysis to form glutamate (L-Glu) and ammonia using a catalytic triad of hCys84, hHis178, and hGlu180 (h and f prefixes indicate if the residue belongs to HisH or HisF, respectively), characterizing it as a class-I GAT. The free ammonia is shuttled across the dimer interface to react with N 1 -(5'-phosphoribosyl)-formimino-5aminoimidazole-4 carboxamide ribonucleotide (PrFAR) in the HisF active site over 25 Å away to form imidazole glycerol phosphate (IGP) and 5'-(5-aminoimidazole-4-carboxamide) ribonucleotide (AICAR). Binding of PrFAR to HisF elicits a V-type allosteric effect that enhances glutamine hydrolysis efficiency in HisH by approximately 4500-fold, primarily influencing the rate of glutamine turnover. The hydrolysis reaction mechanism has not yet been studied at the atomic level in IGPS, to the best of our knowledge. However, similar well-studied enzymes, such as other class-I GATs and cysteine/serine proteases provide a useful theoretical foundation. These reactions are proposed to occur via two stages: acylation and deacylation. In the acylation stage, the nucleophilic hCys84 attacks glutamine to form a glutamyl thioester, first structurally observed in another class-I GAT, carbamoyl phosphate synthetase, via a tetrahedral oxyanion intermediate. In the deacylation stage, nucleophilic attack by a water molecule breaks down the covalent enzyme-substrate intermediate thioester, to yield the glutamate product. The rate-limiting step is proposed to occur during acylation in IGPS, as well as related class-I GATs, carbamyl phosphate synthetase, and anthranilate synthase. IGPS serves as a paradigmatic allosteric enzyme for experimental and methodological developments. An abundance of dynamical information contribute to our understanding of the allosteric mechanism in IGPS at the molecular level. A sequence of residues (hPro49-hGly50-hVal51-hGly52 in IGPS) comprise the oxyanion strand, a structural motif common to class-I GATs that positions a backbone amide hydrogen to stabilize the formation of an oxyanion throughout the reaction. Increased flexibility in the IGPS oxyanion strand upon PrFAR binding was observed in NMR experiments and molecular dynamics (MD) simulations, supporting its mechanistic involvement. An interfacial hydrogen bond between fPro10 and the backbone N-H of hVal51 is weakened in MD simulations with PrFAR, and explains the enhanced flexibility of the oxyanion strand observed in NMR experiments. PrFAR also reduces the opening angle of the interface, which is expected to influence the hydrolysis reaction, although it is unclear if there is a direct effect on the reaction mechanism. A leading hypothesis to explain the allosteric rate effect in IGPS is that PrFAR enables a conformational change in the hVal51 backbone that lowers the rate of glutamine hydrolysis via a catalytically competent oxyanion hole. Contrary to this hypothesis, the hVal51 amide C=O group points into the active site and the N-H away in all but one crystallographic conformations of IGPS in various ligand bound states. The anamolous conformation is observed in a recently deposited structure 7ac8, chains E and F with bound allosteric ligand and Gln substrate. The catalytic hCys84 was mutated to alanine in this structure to disable glutamine turnover and capture IGPS in the presumably active conformation. Osuna and coworkers provide additional support for this hypothesis through MD simulations employing a biasing potential to sample the hVal51 dihedral flip transition. The energetic barrier of this process was estimated to be lower in simulations with PrFAR present (approx. 8 kcal/mol) compared to without PrFAR present (approx. 22 kcal/mol). Exploration of an alternative activation mechanism is warranted for a few reasons. Extensive (10 μs) unbiased MD simulations of IGPS only reproduced the hVal51 dihedral transition when neither Gln nor PrFAR were present, contrary to what was expected. The authors noted the novel IGPS conformation revealed in the 7ac8 crystallographic model could be an artifact of the loss of function hCys84Ala mutation rather than intrinsic to the allosteric mechanism. An alternative activation mechanism that has yet to be evaluated in IGPS involves repositioning of the oxyanion strand relative to the Gln substrate. This activation hypothesis has been proposed for another class-I GAT, aminodeoxychorismate synthase (ADCS) since the presence of two prolines in its oxyanion strand (Pro51-Gly52-Pro53) inhibits Gly52 backbone rotation. All Gln bound IGPS structures show a hydrogen bond between the hGly52 N-H and Gln carbonyl; however, Gln is presumed to be too far from hCys84 to facilitate the nucleophilic attack. The increased oxyanion strand flexibility observed in the presence of PrFAR could facilitate the oxyanion strand reorganization necessary to stabilize the substrate after nucleophilic attack. Despite various X-ray structures, mutagenesis studies, kinetic experiments, and MD simulations, a connection between the allosteric effect and the glutamine hydrolysis mechanism remains hypothetical. This work targets two essential questions regarding IGPS activation: How do local structural aspects of the active site influence the reaction energetics? Is the hVal51 backbone flip required for rate enhancement? To address these questions, we present a dispersion-corrected Density Functional Theory (DFT) study on large (237 atoms) active site cluster models of IGPS in various active and inactive conformations. The multistep reaction leading to thioester formation and ammonia production is investigated and energy decomposition analyses are performed to evaluate the relationship between active site geometry and reaction stabilization. Our results are valuable for future endeavors in modeling the allosteric behavior of this prototypical system by providing clear insights into the atomistic changes required for rate enhancement. Although rigorous workflows have been devised to consider active site multi-state effects on catalysis, this work affords a simplified approach given the available crystallographic data and scope of knowledge from preceding investigations. In doing so, our application of the quantum chemical cluster approach to investigate an allosteric effect and compare the catalytic impact of active site conformational change traverses a challenge in the field, as recently noted by Himo and de Visser.