Geometry optimizations were performed using Gaussian 16, Revision C.01 with the range-separated, dispersioncorrected ωB97X-D 64 functional using the 6-31G* basis set for all C, H, and N atoms and the 6-31+G* 66 basis set for S and O atoms. The α-carbons of selected protein residues were frozen during optimizations to conserve the active site geometry in the absence of the greater protein environment. All geometries were inspected for structural distortions that could have occurred during optimization. Vibrational frequencies were computed to confirm the nature of minima (all real normal modes) and transition structures (a single imaginary normal mode). Stationary point electronic energies were further refined at the B3LYP-D3(BJ)/6-311+G(2d,2p) level of theory and the default IEF-PCM implicit solvent method with internal parameters for diethyl ether (ε=4.24). This procedure is commonly applied in enzymatic QM cluster modeling. The GoodVibes software was used to generate electronic energy profiles. The energetic span between the lowest energy intermediate and the highest energy transition structure (TS) was used to estimate the activation energy. Due to the necessary frozen constraints during geometry optimizations, the vibrational partition function is not considered reliable. Therefore, we focus on the electronic energy profile (i.e., rather than Gibbs energy) throughout. This approximation is common in truncated enzyme modeling and has shown success in mechanistic investigations. The oxyanion hole strength in TS2, TS3, and TS4 of every model was assessed using the Natural Bond Orbital (NBO) analysis program (version 7.0.5) . NBO analysis transforms the optimized atomic orbital basis set into a localized, ideal Lewis structure basis. Second-order perturbation theory analysis of the Fock matrix in the NBO basis is used to estimate the stabilization energy associated with electron delocalization from a filled donor NBO to an unfilled acceptor NBO. The default interaction energy minimum threshold of 0.05 kcal/mol was used. The oxyanion hole interaction energies were investigated between Gln Oε lone pair electrons and the σ* orbitals of the backbone N-H of Leu85, Gly52, and Val51. Additionally, the Val51 Cα-H was investigated in Inactive Val51 and Inactive. To better understand how fGln123 influences the pathway energetics, we evaluated the interaction energy between the fGln123 molecular fragment and the rest of the active site residues for the Active, Inactive fGln123, Inactive Val51 and Inactive models. Absolutely Localized Molecular Orbitals based Energy Decomposition Analysis (ALMO-EDA) implemented in Q-Chem 5.4 was performed at the B3LYP-D3(BJ)/6-311+G(2d,2p) level of theory and CPCM with ε=4.24, to be consistent with the single-point energies used to produce the reaction profiles. Within this framework, the interaction energy is defined as: ∆EINT = ∆EFRZ + ∆EPOL + ∆ECT. EPOL and ECT refer to the polarization and charge transfer energy components, respectively. The EFRZ term combines the interaction energy contributions of the unrelaxed fragment densities, namely attractive dispersion (EDISP), repulsive Pauli (EPAULI) and permanent electrostatics (EELEC): ∆EFRZ = ∆ EDISP + ∆EPAULI + ∆EELEC.