Table IV.
Kinetic Parameters for the Wild-type and the Y76F, H181N, and E224Q Mutant Proteins
| Wild-type | Y76F | H181N | E224Q | |
|---|---|---|---|---|
| Vmax (μM min−1) | 0.37 ± 0.01 | 0.0052 ± 0.0002 | 0.019 ± 0.001 | 0.0049 ± 0.0001 |
| Km for dTDP–sugar (μM) | 4.3 ± 0.2 | 4.6 ± 0.2 | 6.8 ± 1.0 | 3.7 ± 0.2 |
| Km for SAM (μM) | 180 ± 30 | 240 ± 30 | 160 ± 20 | 95 ± 20 |
| kcat (min−1) | 1.70 ± 0.05 | 0.012 ± 0.001 | 0.044 ± 0.002 | 0.011 ± 0.001 |
| kcat/Km for dTDP–sugar (M−1 min−1) | 4.0 × 105 | 2.6 × 103 | 6.5 × 103 | 3.0 × 103 |
| kcat/Km for SAM (M−1 min−1) | 9.4 × 103 | 5.0 × 101 | 2.8 × 102 | 1.2 × 102 |
Abbreviations: dTMP, thymidine monophosphate; dTDP, thymidine diphosphate; ESI, electrospray ionization; HEPES, N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid; HEPPS, N-2-hydroxyethylpiperazine-N′-3-propanesulfonic acid; HPLC, high-performance liquid chromatography; NADPH, nicotinamide adenine dinucleotide phosphate.