The chemical reaction mechanism for TIM-catalyzed deprotonation of the a-carbonyl carbon is not significantly different from that for the non-enzymatic deprotonation of carbon in water, except that the catalytic side chains are tethered at a protein binding pocket which is complementary to the transition state for carbon deprotonation. These side chains are activated in some way for catalysis at the enzyme active site, compared with water: this activation results from the utilization of the phosphodianion binding energy to drive conversion of the flexible open enzyme to the reactive caged enzyme-substrate complex. The results from EVB calculations show that cage formation provides a global thermodynamic activation for substrate deprotonation that corresponds to 13.8 and 13.4 kcal mol -1 increases, respectively, in the driving force for proton transfer from bound DHAP and GAP, and that ca. 75% of this thermodynamic activation (10.4 and 10.3 kcal mol -1 respectively) is expressed at the transition state for substrate deprotonation (Table ). Figure shows that a large number of active site side chains at TIM play a common role in catalysis of the isomerization reactions of DHAP and GAP of stabilizing the enzyme-bound enediolate phosphate relative to bound substrate.