(A) Overview of the mechanisms for the TIM-catalyzed deprotonation of the full substrate GAP and the substrate fragments glycoaldehyde and phosphite dianion (GA·HP_i). (B) A comparison of calculated activation (ΔG^‡_calc, dark blue) and reaction (ΔG⁰_calc, light blue) free energies for the non-enzymatic deprotonation of the full substrate GAP, as well as the corresponding TIM-catalyzed deprotonation of substrates glycoaldehyde (GA), GA·HP_i and GAP. All energies are shown in kcal·mol⁻¹. (C) Population densities of the conformational space sampled by the substrate fragments (C,F) GA and (D,G) GA·HP_i, as well as (E,H) the full substrate GAP, at (C–E) the Michaelis complexes and (F–H) transition states for the TIM-catalyzed deprotonation of these substrates, obtained from EVB simulations, performed as described in ref. [30]. The data on the x- and y-axes based on distances between the donor carbon atoms of the relevant substrate and the backbone α-amino acid carbon atoms of D111 in Chain B and I19 in Chain A. For full details of the analysis see ref. [30]. This figure is adapted from and based on data presented in ref. [30] (https://pubs.acs.org/doi/10.1021/jacs.8b00251), and is reproduced with permission from the American Chemical Society. Please note that requests for permissions regarding further reuse of this figure should be directed to the American Chemical Society.