Figure 5.

Free energy profiles for turnover of GA by free TIM (E_O) and by TIM that is saturated with HPO₃^2- (E_C•HPO₃^2-) constructed using the kinetic parameters reported in Table 5. The profiles show the activation free energy changes calculated using the Eyring equation at 298 K for reactions catalyzed by wildtype and mutant forms of TbbTIM. (A) Reactions catalyzed by wildtype TbbTIM. The difference between the total intrinsic phosphite binding energy of -6.4 kcal/mol and ΔG° = -2.3 kcal/mol for binding of HPO₃^2- to the inactive open enzyme E_O to give the active closed liganded enzyme E_C•HPO₃^2- is attributed to ΔG_C = 4.1 kcal/mol for the conformational change that converts E_O to E_C. (B) Reactions catalyzed by I172A mutant TbbTIM. The overall barrier for conversion of E_C•HPO₃^2- to the transition state for reaction of GA is 3.3 kcal/mol higher than for the reaction catalyzed by wildtype TbbTIM. The green bars show the uncertainty in the barrier to the unactivated reaction of GA, that was drawn using the upper limit of (k_cat/K_m)_E < 0.003 M^-1 s^-1 (Table 5) and a lower limit calculated with the assumption that the total 6.4 kcal/mol intrinsic phosphate dianion binding energy is equal to that for wildtype TbbTIM. (c) Reactions catalyzed by L232A mutant TbbTIM. The red bars show the proposed effect of the L232A mutation on the barrier for the conformational change from E_O to E_C (ΔΔG_C). A comparison of the reaction profiles for wildtype TIM (upper dashed lines) and the L232A mutant (lower dashed lines) shows the effect of this change in ΔG_C on the kinetic parameters for the reaction of the substrate pieces.