TABLE 3.
Kinetics of alarmone and cofactor hydrolysis by XccSAHa
| Protein | Substrate | Vmax (μM/min) | Km (μM) | kcat (s−1) | kcat/Km (s−1 M−1) |
|---|---|---|---|---|---|
| Alarmone | pGpp | 4.89 ± 0.22 | 19.73 ± 4.75 | 1.63 ± 0.07 | (0.86 ± 0.18) × 105 |
| ppGpp | 20.96 ± 0.44 | 14.13 ± 2.02 | 6.99 ± 0.15 | (5.00 ± 0.82) × 105 | |
| pppGpp | 42.89 ± 4.36 | 29.14 ± 4.41 | 14.30 ± 1.45 | (4.93 ± 0.25) × 105 | |
| pppApp | 73.12 ± 4.49 | 45.82 ± 3.87 | 48.75 ± 2.99 | (10.67 ± 0.75) × 105 | |
| Cofactor | NADPH | 2.88 ± 0.26 | 132.2 ± 39.09 | 0.048 ± 0.004 | (3.63 ± 0.20) × 102 |
a
Calculations for SAH are based on data presented in Fig. 2 and 3. Vmax and Km were calculated by nonlinear regression according to the Michaelis-Menten equation. Alarmone hydrolysis was measured from hydrolysis of 32P-labeled alarmones, while NADPH hydrolysis was measured from inorganic phosphate release using a malachite green assay. XccSAH was used at 25 nM for pppApp, 50 nM for (pp)pGpp, and 1 μM for NAPDH. Each value represents the mean and standard deviation from three independent experiments.