Table 3.
Kinetic Data for the Reaction of [1-13C]-GA Catalyzed by Wildtype Tbb TIM in D2O in the Absence and Presence of Phosphite Dianion.a
| [HPO32−] (M) | [TIM] (M) | kobs (s−1)b | (kcat/Km)obs (M−1s−1)c |
|---|---|---|---|
| 0 | 2.72 × 10−4 | 2.15 × 10−6 | 0.13 d 0.07e |
| 2.6 × 10−3 | 1.36 × 10−4 | 7.08 × 10−5 | 8.6 |
| 5.3 × 10−3 | 1.15 × 10−4 | 1.01 × 10−4 | 14.3 |
| 7.9 × 10−3 | 1.02 × 10−4 | 1.13 × 10−4 | 18.2 |
| 9.9 × 10−3 | 9.50 × 10−5 | 1.28 × 10−4 | 22.0 |
| 12.6 × 10−3 | 9.50 × 10−5 | 1.56 × 10−4 | 26.9 |
| 15.3 × 10−3 | 8.20 × 10−5 | 1.48 × 10−4 | 29.6 |
| 17.3 × 10−3 | 6.82 × 10−5 | 1.26 × 10−4 | 30.3 |
For reactions of 20 mM [1-13C]-GA in 20 mM imidazole (20% free base), pD 7.0, 25 °C, and I = 0.1 (NaCl).
Observed first-order rate constant for the reaction of [1-13C]-GA calculated using eq 1.
Observed second-order rate constant for the TIM-catalyzed reaction of [1-13C]-GA calculated using eq 2, unless noted otherwise.
Second-order rate constant determined by monitoring the disappearance of total [1-13C]-GA.
Second-order rate constant for the reactions of [1-13C]-GA at the TIM active site, calculated from the overall reaction rate constant of 0.13 M−1 s−1 (determined from two independent experiments) and the 52% product yield (Table 4).