Table 1. Activation Barriers and Reaction Free Energies for the Deprotonation of DHAP and GAP by Different TIM Variants, To Form Enediolate Phosphate Reaction Intermediatesa.
| substrate | catalyst | ΔGexp⧧ | ΔGcalc⧧ | ΔGTIM⧧ – ΔGnon | ΔGWT⧧ – ΔGmut | ΔGcalc‡ – ΔGexp | ΔG°calc | ΔG°TIM – ΔG°non |
|---|---|---|---|---|---|---|---|---|
| DHAP | CH3CH2CO2– | 25.4 ± 0.03 | 19.0 ± 0.03 | |||||
| WT-TIM | 13.7b,c | 15.0 ± 0.4 | –10.4 | 1.3 | 5.2 ± 0.4 | –13.8 | ||
| I170A | 15.8b | 15.6 ± 0.5 | –9.8 | –0.6 | –0.2 | 5.8 ± 0.7 | –13.2 | |
| L230A | 16.6b | 15.8 ± 0.3 | –9.6 | –0.8 | –0.8 | 7.0 ± 0.4 | –12.0 | |
| I170A/L230A | 17.4b | 16.8 ± 0.2 | –8.6 | –1.8 | –0.6 | 7.5 ± 0.3 | –11.5 | |
| K12G | 18.7 ± 0.3 | –6.7 | –3.7 | 10.5 ± 0.5 | –8.5 | |||
| E97A | 13.9b (14.1c) | 14.8 ± 0.4 | –10.6 | 0.2 | –0.9 | 4.8 ± 0.5 | –14.2 | |
| K12G/E97A | 18.9 ± 0.3 | –6.5 | –3.9 | 10.6 ± 0.4 | –8.4 | |||
| E97D | 13.9b,c | 14.9 ± 0.3 | –10.5 | 0.1 | 1.0 | 4.0 ± 0.3 | –15.0 | |
| E97Q | 14.1b (14.5c) | 14.9 ± 0.3 | –10.5 | 0.1 | 0.8 | 5.2 ± 0.6 | –13.8 | |
| P166A | 16.3b | 17.2 ± 0.3 | –8.2 | –2.2 | 0.1 | 8.5 ± 0.3 | –10.5 | |
| GAP | CH3CH2CO2– | 24.0 ± 0.03 | 16.0 ± 0.04 | |||||
| WT-TIM | 12.9b (12.5d) | 13.7 ± 0.5 | –10.3 | 0.8 | 2.6 ± 0.6 | –13.4 | ||
| I170A | 16.0b | 15.5 ± 0.7 | –8.5 | –1.8 | –0.5 | 4.1 ± 0.7 | –11.9 | |
| L230A | 14.2b | 14.4 ± 0.3 | –9.6 | –0.7 | 0.2 | 3.3 ± 0.4 | –12.7 | |
| I170A/L230A | 16.3b | 15.2 ± 0.3 | –8.8 | –1.5 | –1.1 | 4.3 ± 0.5 | –11.6 | |
| K12G | 17.7c | 16.2 ± 0.4 | –7.8 | –2.5 | –1.5 | 6.0 ± 0.4 | –10.0 | |
| E97A | 13.7 ± 0.7 | –10.3 | 0.0 | 1.7 ± 0.7 | –14.3 | |||
| K12G/E97A | 16.5 ± 0.4 | –7.5 | –2.8 | 6.5 ± 0.4 | –9.5 | |||
| E97D | 15.2d | 13.0 ± 0.4 | –11.0 | 0.7 | –2.2 | 1.5 ± 0.6 | –14.5 | |
| E97Q | 17.4d | 14.4 ± 0.4 | –9.6 | –0.7 | –3.0 | 3.4 ± 0.4 | –12.6 | |
| P166A | 15.6b | 16.4 ± 0.4 | –7.6 | –2.7 | 0.1 | 5.4 ± 0.4 | –10.6 |
All energies are shown in kcal mol–1. Calculated energies are averages and standard error of the mean over 30 individual EVB trajectories, as described in the Methodology section. Shown here are both the values calculated in aqueous solution (CH3CH2CO2– as the general base) and those in different TIM variants. The corresponding experimental values are based on kinetic data for different TIM variants.
Data from TbbTIM.
Data from yTIM.
Data from PfTIM, where available, as presented in refs (16), (40)–44, (50), and (70). ΔG⧧calc and ΔG⧧exp denote the calculated and experimental activation free energies, respectively, ΔG0calc denotes the calculated reaction free energy, ΔG⧧TIM – ΔG⧧non denotes the difference between the activation free energy calculated for the TIM-catalyzed reaction and for the background reaction in aqueous solution, ΔG⧧WT – ΔG⧧mut denotes the difference between the activation free energy calculated for wild-type TIM and for the different TIM variants considered in this work, ΔG⧧calc – ΔG⧧exp denotes the difference between the calculated and experimental activation free energies for wild-type TIM and the different TIM variants considered in this work, and ΔG0TIM – ΔG0non denotes the difference between the reaction free energy calculated for different TIM variants and for the background reaction in aqueous solution.