Table 2.
Kinetic properties of PpAOS2 with different hydroperoxy fatty acid substrates
| Substrate | KM[μM] | Vmax[μM/min] | kcat[1/min] | kcat/KM[min-1M-1× 106] |
|---|---|---|---|---|
| 9-HPOD |
36 +/− 5 |
0.02 +/− 0.001 |
5 |
0.14 |
| 9- HPOT(n-3) |
40 +/− 4 |
0.01 +/− 0.001 |
2.5 |
0.06 |
| 9- HPOT(n-6) |
28 +/− 4. |
0.03 +/− 0.002 |
7.5 |
0.27 |
| 13-HPOD |
27+/− 3 |
0.04 +/− 0.002 |
10 |
0.37 |
| 13- HPOT(n-3) |
30 +/− 5 |
0.02 +/− 0.002 |
5 |
0.17 |
| 13- HPOT(n-6) |
42 +/− 10 |
0.02 +/− 0.002 |
5 |
0.12 |
| 12-HPETE | 10 +/− 5 | 0.49 +/− 0.057 | 12250 | 1228.69 |
Kinetic properties were determined by measuring the initial time-dependent substrate consumption at 234 nm at different substrate concentrations typically ranging from 2–100 μM. For analysis between 20 and 30 data points data points were fitted to the Michaelis-Menten equation. Note that the PpAOS2-concentration used for incubations with 12-HPETE was different (1 nM) from those used for incubations with the other substrates (100 nM). Kcat values are corrected to 100% heme occupancy from the ~4% heme content in the enzyme preparation.