Table 1.
Observed relaxation rates (inverse of relaxation times) for a 23 to 30 °C T-jump for the ternary complexes of bsLDH purified from the wild type and the single-tryptophan mutants.
| Source of bsLDH | NADH fluorescence T-jump trace | Tryptophan fluorescence T-jump trace | |||
|---|---|---|---|---|---|
|
| |||||
| 1 / t1, s−1 | 1 / t2, s−1 | 1 / t3, s−1 | 1 / t1, s−1 | 1 / t2, s−1 | |
|
| |||||
| Wild type | 327 ±20; 78% | 1173 ±500; 8% | 45800 ±5000; 13% | not measured | not measured |
| G106W | not measured | not measured | not measured | 320 ±230; 76% | 2300 ±1000; 24% |
| Y190W | 303 ±11; 56% | 3500 ±1900; 29% | 35500 ±2500; 15% | 913 ± 18; 74% | 16400 ±800; 26% |
| Y248W | 210 ±19; 85% | 2900 ±2400; 5% | 26000 ±7000; 10% | 480 ±40 | |
| Y279W | 460 ±22; 76% | 4100 ±3500; 7% | 19000 ±5000; 17% | 434 ± 13 | |
Most ternary mixtures contain 80 μM bsLDH, 80 μM NADH and 1200 μM oxamate in TEA buffer at pH 6.0. The only exception was for G106W, where similar mixtures were prepared in TEA buffer of pH 7.2. For the G106W, T-jump was from 15 to 24 °C. Percent contribution of each component to the amplitude follows each relaxation rates. For comparison, T-jump IR studies27 measuring the modulation of the C2=O stretch mode of oxamate in the ternary complex of bsLDH found two transients at 490 and 1461 s−1 (at 22 °C).