The empirical valence bond (EVB) approach was used in this work to model the TIMcatalyzed deprotonation of substrates DHAP and GAP, in wild-type and substituted TIM variants. We selected this approach based on our previous successes when using this model to capture the catalytic effect of wild-type TIM as well as of key active site substitutions on the rate-limiting deprotonation of these substrates. All simulations in this study were performed using the OPLS-AA force field as implemented in the Q6 version of the Q simulation package, following the protocol outlined in detail in our previous studies. Our starting point for all EVB simulations was the crystal structure of wild-type yeast TIM (yTIM) in complex with DHAP (PDB ID: 1NEY ). In the case of the TIM variants studied in this work, all glycine and alanine substitutions were generated by simple truncation of the appropriate side chains, whereas in the case of the E97D and E97Q substitutions, the rotamers for the aspartate and glutamine side chains, respectively, were selected from the Dunbrack 2010 backboneindependent rotamer library, as implemented into Chimera. Simulations were performed on wild-type TIM, as well as on the K12G, E97A, K12G/E97A, E97D, E97Q, P166A, I170A, L230A and I170A/L230A variants.