Table 1. Activation (ΔG^⧧) and Gibbs Free Energies (ΔG°) for the Deprotonation of DHAP and GAP by Wild-Type and Mutant Forms of TIM, To Form Enediolate Phosphate Reaction Intermediates^a.

substrate	catalyst	ΔGcalc⧧b	ΔGcalc⧧ – ΔGexpc	ΔGTIM⧧ – ΔGnond	ΔGWT⧧ – ΔGmute	ΔGcalc°f	ΔGTIM⧧ – ΔGnong
DHAP	CH3CH2CO2– in water	25.2 ± 0.2				18.9 ± 0.2
	WT-TIM	14.5 ± 1.4	0.4	–10.7		5.6 ± 1.8	–13.3
	I170A	16.3 ± 1.5	0.5	–8.9	–1.8	7.6 ± 1.4	–11.3
	L230A	16.7 ± 0.8	0.1	–8.5	–2.2	8.6 ± 0.8	–10.3
	I170A/L230A	18.5 ± 1.0	1.1	–6.7	–4.0	11.0 ± 1.3	–7.9
GAP	CH3CH2CO2– in water	24.1 ± 0.2				16.1 ± 0.2
	WT-TIM	12.9 ± 0.8	0.0	–11.2		2.5 ± 0.9	–13.6
	I170A	16.2 ± 1.7	0.2	–7.9	–3.3	5.7 ± 1.9	–10.4
	L230A	14.9 ± 0.8	0.7	–9.2	–2.0	3.1 ± 1.0	–13.0
	I170A/L230A	16.5 ± 1.4	0.2	–7.6	–3.6	5.4 ± 1.8	–10.7

^aAll energies are shown in kcal mol^–1.

^bActivation barrier for proton transfer from the carbon acid substrate to a carboxylate base, for reactions in water or at the active site of yTIM. The calculated energies and standard deviations are obtained as the average of 30 independent EVB trajectories/system (Methodology).

^cDifference between the calculated activation barriers for proton transfer and the barriers determined by experiment reported in Table S2.

^dDifference between the activation barriers for proton transfer at TIM and in aqueous solution.

^eDifference between the activation barriers for proton transfer at wild-type and the specified mutant TIM.

^fCalculated change in Gibbs free energy for proton transfer from the carbon acid substrate to a carboxylate base in water or at the active site of yTIM.

^gThe difference in ΔG_calc^° for proton transfer at TIM (G_TIM) and in aqueous solution (G_non^°).