Comparison of anandamide metabolism by wild-type and mutant CYP2B6 enzymes
CYP2B6 enzymes were reconstituted with reductase, and the kinetic data were determined as described under Materials and Methods. The concentrations of anandamide ranged from 0.75 to 10 μM. Metabolites were extracted and analyzed by ESI-LC-MS. The data represent the mean of experiments done in triplicate. The data were fitted to the Michaelis-Menton model using Prism software to derive the kinetic constants.
| . | 20-HETE-EA | 14,15-EET-EA | 11,12-EET-EA | 8,9-EET-EA | 5,6-EET-EA |
|---|---|---|---|---|---|
| Km (μM) | |||||
| CYP2B6.1 (WT) | N.D. | 3.6 | 1.320 | 1.210 | 1.320 |
| CYP2B6.4 | 0.362 | 1.237 | 1.380 | 1.510 | 0.591 |
| CYP2B6.6 | N.D. | 1.501 | 1.341 | 1.211 | N.D. |
| CYP2B6.9 | N.D. | N.D. | 3.089 | N.D. | N.D. |
| Vmax (pmol · min−1 · pmol−1) | |||||
| CYP2B6.1 (WT) | N.D. | 2.040 | 7.560 | 3.120 | 4.596 |
| CYP2B6.4 | 0.546 | 1.721 | 10.450 | 4.450 | 4.551 |
| CYP2B6.6 | N.D. | 1.062 | 2.221 | 0.693 | N.D. |
| CYP2B6.9 | N.D. | N.D. | 5.549 | N.D. | N.D. |
WT, wild-type; N.D., not detected.