Table 2.
Steady-state kinetics
| Nitro compound | kcat, s−1 | Km(NADPH), μM | Km(nitro), μM |
|---|---|---|---|
| Nitrocyclohexene | 4.4 ± 0.2 | 12 ± 2 | 10 ± 1 |
| Nitrostyrene | 5.0 ± 0.1 | ND | 4.2 ± 0.3 |
| Nitrovinylthiophene | 4.3 ± 0.2 | ND | 7.7 ± 0.5 |
The kinetic constants were obtained from enzyme-monitored turnover experiments carried out in 0.05 M KPi buffer, pH 7.0, 25°C, and analyzed as described (11). The Km(NADPH) values with nitrostyrene and nitrovinylthiophene were in the low μM region, but could not be determined accurately because of the overlapping spectra of the nitrocompounds with that of the NADPH. In the case of nitrocyclohexene there was essentially no overlap, and the Km(NADPH) was determined from analysis of the absorbance changes at 340 nm.