The overall agreement between the experimental and computed activation barriers is generally within ± 1 kcal mol -1 , and is similar to the previously published agreement between experimentally and computationally determined effects, respectively, of I170A, L230A, and I170A/L230A substitutions on the activation barriers for TbbTIM-and yTIM-catalyzed deprotonation of GAP and DHAP. We have increased the radius of the water droplet/mobile region in our EVB simulations from 20 Å in our previous computational studies of TIM and its variants to 24 Å in the present work. This improves the description of TIM flexibility through the use of a larger mobile region, but comes with a greater computational cost associated with the increased size of the water droplet. The 4 Å increase in the radius of the water droplet (and thus also the size of the mobile region in the EVB simulations) in the present study results in up to a 0.8 kcal mol -1 higher calculated activation barriers for the deprotonation of both DHAP and GAP. However, there is no discernible improvement in the agreement between the experimental and calculated activation barriers for wild-type TIM and the I170A, L230A