Table 4.
Initial rate kinetics for HpxL and PcxL
Rates were determined by monitoring NADPH consumption via UV-visible spectrophotometer. Vmax, Km, and KI were determined by fitting the data with either the Michaelis-Menten (MM) or substrate inhibition (SI) model. If the data were fitted to the MM model, a KI was not applicable (NA). Only the best fit is shown. Reaction components included: 50 mm sodium phosphate buffer, pH 7.8, 300 μm NADPH, 50 μm FAD, substrate ranging from 62.5 μm to 150 mm, and enzyme concentrations of 1 μm or 5 μm for PcxL and 5 μm or 10 μm for HpxL. The substrates used were 2AEPn (Sigma-Aldrich), synthetic (R)-1H2AEPn, and synthetic (S)-1H2AEPn. This experiment was performed in triplicate for each curve.
| Enzyme | Substrate | Kinetic model | Vmax | Apparent Km | kcat/Km | KI |
|---|---|---|---|---|---|---|
| s−1 | mm | m−1 s−1 | mm | |||
| PcxL | 2AEPn | MM | 1.22 ± 0.02 | 3.3 ± 0.5 | 370 | NA |
| (R)-1H2AEPn | MM | 0.39 ± 0.01 | 0.48 ± 0.02 | 810 | NA | |
| (S)-1H2AEPn | MM | 0.32 ± 0.03 | 6 ± 1 | 53 | NA | |
| HpxL | 2AEPn | MM | 0.032 ± 0.002 | 4.3 ± 0.8 | 7.4 | NA |
| (R)-1H2AEPn | SI | 0.06 ± 0.03 | 4 ± 2 | 15 | 2 ± 1 | |
| (S)-1H2AEPn | MM | 0.039 ± 0.001 | 0.39 ± 0.03 | 100 | NA |