Table 3. Localization of the Effects of Mutations of yTIM on the Catalyzed Reactions of the Substrate Pieces, Determined As Described in the Text.

		[(kcat/Km)E]obsb
mutant TIM	intrinsic dianion binding energy (−RT ln(Kd⧧))a	(kcat/Km)E′c	KCd
Y208T	↓	no change	no change
Y208S	↓	no change	no change
Y208A	↓	no change	no change
Y208F	↓	↓	no change
S211A	no change	no change	↓
S211G	small decreasee	no change	no change
Y208T/S211G	↓	small increase	no change
208-TGAG for 208-YGGS (LRM)f	small decreasee	↓	no change
P168Ag	no change	↓	no change

^aThe intrinsic dianion binding energy, calculated from data reported in Table 2 using eq 6 derived for Scheme 7.

^bThe observed rate constant for enzyme-catalyzed reactions of the truncated substrate glycolaldehyde; [(k_cat/K_m)_E]_obs = (k_cat/K_m)_E′(K_C) where (k_cat/K_m)_E′ is the second-order rate constant for the reactions of glycolaldehyde catalyzed by the closed form of TIM (Scheme 6). It is assumed when there is no change in [(k_cat/K_m)_E]_obs for a mutant enzyme, that there is no effect of the mutation on either (k_cat/K_m)_E′ or (K_C).

^cThe true second-order rate constant for the reactions of glycolaldehyde catalyzed by the closed form of TIM (Scheme 6).

^dThe equilibrium constant for conversion of the inactive open form of TIM (E_O) to the active closed enzyme (E_C, Scheme 6).

^eAn approximate 0.4 kcal/mol decrease.

^fLoop replacement mutation of TIM from chicken muscle.^29,82

^gThe P168A mutation of TIM from T. brucei.(29,83)