Table 1.
Apparent kinetic parameters for the proteolysis of Boc-QAR-AMC by Der p 1 in the presence of GSTpi, using GSH and l-Cys as alternative reducing agents.
| GSTpi (μM) |
0 | 0.15 | 0.3 | 0.6 | 1.3 | 2.5 | |
|---|---|---|---|---|---|---|---|
| (GSH) | |||||||
| Hanes-Woolf | Vmax (nM/min) | 35.8 | 50.3 | 51.4 | 54 | 54.3 | 62.1 |
| Km (mM) | 6.2 | 4.5 | 2.3 | 1.8 | 0.6 | 0.4 | |
| Kcat/Km (min−1) | 5.8 × 10−6 | 1.1 × 10−5 | 2.2 × 10−5 | 3.0 × 10−5 | 9.1 × 10−5 | 1.4 × 10−4 | |
| Eadie-Hofstee | Vmax (nM/min) | 25.4 | 43.8 | 44.5 | 49.8 | 51.6 | 53.8 |
| Km (mM) | 5.7 | 3.4 | 1.8 | 0.8 | 0.5 | 0.3 | |
| Kcat/Km (min−1) | 4.5 × 10−6 | 1.3 × 10−5 | 2.5 × 10−5 | 6.2 × 10−5 | 1.0 × 10−4 | 1.8 × 10−4 | |
|
| |||||||
| GSTpi (μM) | 0 | 0.15 | 0.3 | 0.6 | 1.3 | 2.5 | |
| (l-Cys) | |||||||
| Hanes-Woolf | Vmax (nM/min) | 72.1 | 82.2 | 103 | 112.2 | 125.4 | 132.6 |
| Km (mM) | 0.4 | 0.3 | 0.3 | 0.2 | 0.2 | 0.2 | |
| Kcat/Km (min−1) | 1.8 × 10−4 | 2.7 × 10−4 | 3.3 × 10−4 | 5.6 × 10−4 | 6.3 × 10−4 | 6.6 × 10−4 | |
| Eadie-Hofstee | Vmax (nM/min) | 66.7 | 78.2 | 89.4 | 103 | 109.7 | 120.7 |
| Km (mM) | 0.2 | 0.2 | 0.2 | 0.1 | 0.1 | 0.1 | |
| Kcat/Km (min−1) | 3.3 × 10−4 | 3.9 × 10−4 | 4.5 × 10−4 | 1.0 × 10−3 | 1.1 × 10−3 | 1.2 × 10−3 | |
The apparent Km, Vmax and Kcat/Km values were determined using both Hanes-Woolf and Eadie-Hofstee plots. GSTpi (0–2.5 μM) and substrate (75 μM) concentrations were kept constant for each scanning of the reducing agent (from 0.15 to 2.5 mM).