TABLE 2.
Apparent steady-state kinetic parameters of KshA1 and KshA5
Assays were performed using potassium phosphate (I = 0.1 m, pH 7.0) at 22 °C containing 100 μm NADH (∼260 μm O2). Errors in parentheses were calculated from the weighted mean of the error in the enzyme concentration, and the standard errors in the parameters were estimated using LEONORA.
| Substrate | KshA1 |
KshA5 |
|||||
|---|---|---|---|---|---|---|---|
| kcat | Km | kcat/Km | kcat | Kma | kcat/Km | KiS | |
| s−1 | μm | s−1 mm−1 | s−1 | μm | s−1 mm−1 | μm | |
| 5α-Androstan-3,17-dione | 0.8 (0.1) | 6.1 (0.3) | 140 (20) | ||||
| 4-Estren-3,17-dione | 0.5 (0.1) | 400 (200) | 1.2 (0.3) | 0.8 (0.1) | 0.8 (0.1) | 1000 (200) | |
| Testosterone | 1.4 (0.3) | 110 (20) | 13 (2) | 0.8 (0.1) | 0.5 (0.1) | 1500 (300) | 130 (30) |
| ADD | 1.3 (0.2) | 50 (10) | 27 (4) | 0.7 (0.1)b | |||
| 4-BNC | 2.6 (0.5) | 2.2 (0.2) | 1200 (20) | 0.6 (0.1) | 1.2 (0.5) | 500 (200) | 110 (20) |
| 1,4-BNC | 0.9 (0.2) | 1.0 (0.2) | 900 (200) | 0.5 (0.1) | 0.5 (0.3) | 1000 (500) | 40 (20) |
| 1,4-BNC-CoA | 1.1 (0.3) | 2 (1) | 600 (400) | 1.7 (0.6) | 500 (200) | 3.7 (0.8) | |
| O2c | 1.7 (0.3) | 1.5 (0.2) | 1100 (200) | 0.8 (0.1) | 49 (6) | 17 (3) | |
a For parameters calculated using the substrate inhibition equation, the Km values represent Ks values.
b Data were calculated by averaging the maximal rates from Fig. 3.
c Data were determined using 50 μm 4-BNC and 5 μm 4-estren-3,17-dione for KshA1 and KshA5, respectively.