Table 1.
Kinetic Parameters for Hydrolysis of 2′,3′ cAMP by CthPnkp
| Protein | Km (mM) | kcat (min−1) |
|---|---|---|
| Wild-type | 18 ± 1.4 | 536 ± 24 |
| H189A | 29 ± 1 | 87 ± 6 |
| H189D | 20 ± 1 | 2060 ± 480 |
| H189E | 28 ± 2 | 1030 ± 79 |
| D233E | 62 ± 6.5 | 195 ± 43 |
| D236A | 7.8 ± 1.5 | 272 ± 12 |
| D236N | 6.6 ± 0.2 | 346 ± 14 |
| D236E | 18 ± 1.1 | 362 ± 20 |
| R237A | 77 ± 10 | 513 ± 1 |
| R237K | 100 ± 7.5 | 1570 ± 94 |
| R237Q | 60 ± 6.5 | 1210 ± 10 |
| H323A | 32 ± 4.3 | 546 ± 34 |
| H323Q | 24 ± 2.6 | 390 ± 87 |
| H376N | 4.7 ± 0.1 | 308 ± 18 |
| H376D | 3.9 ± 0.7 | 192 ± 46 |
| D392A | 100 ± 2 | 106 ± 4.5 |
| D392E | 18 ± 0.8 | 958 ± 100 |
| D392N | 31 ± 9.1 | 1120 ± 240 |
Reaction mixtures (25 µl) containing 50 mM Tris–HCl (pH 7.5), 0.5 mM MnCl2, 0.1 unit of CIP, increasing concentrations (0.625, 1.25, 2.5, 5, 10, or 20 mM) of 2′,3′ cAMP, and a fixed amount of one of the indicated CthPnkp proteins were incubated for 15 min at 45°C. Pi production was plotted as a function of substrate concentration, and then Km and kcat values were calculated from double-reciprocal plots of the data. The values in the table are averages from two independent substrate titration experiments.