Kinetic parameters for CYP2A13 and CYP2A13 N297A reactions
Values for Km and Vmax represent mean ± S.E. of two to five independent experiments.
Reactiona | CYP2A13 wt Km | CYP2A13 wt Vmax | CYP2A13 N297A Km | CYP2A13 N297A Vmax |
|---|---|---|---|---|
| μMb | pmol/min/pmol P450 | μMb | pmol/min/pmol P450 | |
| Coumarin 7-hydroxylation | 3.7 ± 0.39 | 0.59 ± 0.01 | 16.6 ± 0.9 | 1.94 ± 0.03 |
| Coumarin 3,4-epoxidation | 3.8 ± 0.43 | 0.25 ± 0.006 | 14.8 ± 1.1 | 9.25 ± 0.09 |
| Couamarin 3-hydroxylation | 6.3 ± 1.5 | 0.06 ± 0.003 | 18.9 ± 0.3 | 1.12 ± 0.03 |
| NNK α-hydroxylationc | 11.9 ± 3.2 | 0.85 ± 0.07 | 66.7 ± 8.1 | 0.87 ± 0.09 |
| (S)-NNN 5′-hydroxylation | 30.7 ± 10.1 | 1.28 ± 0.11 | 108 ± 22.1 | 1.12 ± 0.12 |
↵ a Coumarin concentrations were 1, 2, 5, 10, 20, 40, 100, 200, and 500 μM; NNK concentrations were 0.5, 2, 5, 10, 25, 50, 75, 100, 200, and 300 μM; and (S)-NNN concentrations were 10, 25, 50, 100, 250, 500, 750, and 1000 μM. Products were detected by UV-HPLC (coumarin) or radioflow-HPLC (NNK and NNN). Details are described under Materials and Methods.
↵ b Km values for all substrates were significantly different for CYP2A13 N297A compared with CYP2A13 (P < 0.03).
↵ c α-Hydroxylation was quantified as the sum of methyl and methylene hydroxylation that occurred in a ratio of 1:3 at all NNK concentrations.