Table 1.
Kinetic Constants for OMP, EO and EO/HPi and Intrinsic Binding Energies of the 5′-Phosphate Group of OMP at pH 7.1 and 25 °C.
| MtOMPDC |
kcat OMP s−1 |
kcat/Km OMP M−1 s−1 |
ΔΔG‡ kcal/mola |
kcat/Km EO M−1 s−1 |
ΔΔG‡ kcal/mola |
(kcat/Km)/K D EO•HPib M−2 s−1 |
ΔΔG‡ kcal/mola |
5′-Phosphate IBEc kcal/mol |
|---|---|---|---|---|---|---|---|---|
| Wild type | 4.6 | 2.9 × 106 | 8.7 × 10−3 | 2500 | 11.6 d | |||
| V182A | 3.4 | 1.4 × 105 | 1.8 | 1.3 × 10−3 | 1.1 | 190 | 1.5 | 10.9 |
| I199A | 3.9 | 9.1 × 105 | 0.7 | 1.9 × 10−3 | 0.9 | 980 | 0.6 | 11.8 |
| V201A | 4.0 | 9.5 × 105 | 0.7 | 3.1 × 10−3 | 0.6 | 690 | 0.8 | 11.5 |
| I218A | 3.3 | 2.8 × 105 | 1.4 | 2.3 × 10−3 | 0.8 | 340 | 1.2 | 11.0 |
| V182A/I199A | 3.1 | 4.9 × 104 | 2.4 | 3.9 × 10−4 | 1.8 | 81 | 2.0 | 11.0 |
| V182A/V201A | 2.5 | 4.9 × 104 | 2.4 | 5.0 × 10−4 | 1.7 | 30 | 2.6 | 10.9 |
a
Calculated from the ratio of the second-order or third-order rate constants for the wild type and mutant enzyme.
b
Third-order rate constant for reaction of EO/HPi.
c
Transition state stabilization by the 5′-phosphate group of OMP, calculated from the ratio of the values of kcat/Km for OMP and EO. IBEs for phosphite dianion can be calculated from the ratio of (kcat/Km)/KD for EOHPi and kcat/Km for EO.