(4) The side chains of I170 and L230, which fold over the catalytic carboxylate side chain of E165 at the closed form of TIM and displace to the bulk aqueous solvent four of six water molecules that lie within 6 Å of this side chain. We previously reported the results of simulations to model the effect of I170A, L230A and I170A/L230A substitutions on the activation barrier for deprotonation of GAP and DHAP bound to yeast TIM (ScTIM), but report here slightly modified barriers obtained by the improved computational methods used in the present work. The I170A and L230A substitutions examine the precision of the EVB method in modelling the effect of a change in the polarity of the enzyme active site through replacement of hydrophobic side chains by solvent water. The EVB simulations do an excellent job of modeling the experimental effect of substitution of both the polar side chains of K12 and E97, and of the substitution of the nonpolar side chains of P166, I170 and L230, on the activation barriers (DG ‡ ) for deprotonation of enzyme-bound DHAP and GAP by the carboxylate side chain of E165. These simulations also provide values of DG o for proton transfer from the bound substrate to TIM to give the enediolate phosphate intermediate and the protonated carboxylic acid side chain, which cannot be determined by experiment. We report here a large extension of previously reported linear free energy relationships between the activation barriers DG ‡ and Gibbs free energy change, DG o , for TIMcatalyzed deprotonation of enzyme-bound DHAP and GAP, to form the corresponding enediolate reaction intermediates, with Brønsted slopes of β = 0.73 (R 2 = 0.994) and β = 0.74 (R 2 = 0.995) for the deprotonation of DHAP and GAP, respectively. This extended Brønsted relationship shows that the substituted polar and nonpolar side chains act cooperatively for the common purpose of reducing the standard-state Gibbs free energy of reaction for proton transfer from substrate to TIM, and that 70% of the change in the driving force for the reaction in water is expressed at the transition state for proton transfer. This change in reaction driving force is obtained through the direct stabilization of the enediolate phosphate by interaction with the cationic K12 side chain, and through the precise placement of the nonpolar P166, I170 and L230 side chains at a substrate cage, engineered by Nature in order to minimize the standard-state Gibbs free energy of reaction for deprotonation of the enzyme bound substrate.