Kinetic parameters of nicotine C-oxidation and coumarin 7-hydroxylation by microsomes from 293FT cells expressing CYP2A6 wild-type and variant proteins
These data represent the mean ± S.D. of three independently performed catalytic assays.
| Variants | Nucleotide Changes | Amino Acid Changes | Nicotine C-Oxidation | Coumarin 7-Hydroxylation | ||||
|---|---|---|---|---|---|---|---|---|
| Km | Vmax | CLint (Vmax/Km) | Km | Vmax | CLint (Vmax/Km) | |||
| μM | pmol/min per pmol CYP2A6 | nl/min per pmol CYP2A6 (% of wild-type) | μM | pmol/min per pmol CYP2A6 | μl/min per pmol CYP2A6 (% of wild-type) | |||
| CYP2A6*1 | 33.3 ± 5.44 | 5.91 ± 1.26 | 176 ± 11.2 | 1.94 ± 0.06 | 5.89 ± 0.34 | 3.04 ± 0.08 | ||
| CYP2A6*2a,c | 1799T>A | Leu160His | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. |
| CYP2A6*5a,c | 6582G>T | Gly479Val | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. |
| CYP2A6*6a,c | 1703G>A | Arg128Gln | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. |
| CYP2A6*7 | 6558T>C | Ile471Thr | 267 ± 59.9 | 2.36 ± 0.41 | 9.14 ± 2.44* (5%) | 4.55 ± 1.94 | 3.33 ± 0.62 | 0.80 ± 0.17* (26%) |
| CYP2A6*8 | 6600G>T | Arg485Leu | 103 ± 16.3 | 2.10 ± 0.24 | 20.6 ± 2.44* (12%) | 3.13 ± 0.30 | 2.22 ± 0.16* | 0.71 ± 0.06*** (23%) |
| CYP2A6*10b,d | 6558T>C | Ile471Thr | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. |
| 6600G>T | Arg485Leu | |||||||
| CYP2A6*11 | 3391T>C | Ser224Pro | 142 ± 31.3 | 4.31 ± 0.34 | 31.6 ± 5.88* (18%) | 3.30 ± 0.37 | 2.57 ± 0.03* | 0.79 ± 0.09*** (26%) |
| CYP2A6*13 | 13G>A | Gly5Arg | 30.8 ± 0.80 | 3.94 ± 0.20 | 128 ± 5.36 (73%) | 1.37 ± 0.03* | 2.61 ± 0.19 | 1.90 ± 0.17 (63%) |
| CYP2A6*14 | 86G>A | Ser29Asn | 28.8 ± 0.65 | 7.01 ± 0.12 | 243 ± 5.67 (138%) | 1.77 ± 0.15 | 8.02 ± 0.43 | 4.53 ± 0.16* (149%) |
| CYP2A6*15 | 2134A>G | Lys194Glu | 23.4 ± 0.74 | 9.03 ± 0.09 | 385 ± 8.46*** (219%) | 1.27 ± 0.03* | 9.10 ± 0.31* | 7.17 ± 0.28* (236%) |
| CYP2A6*16 | 21611C>A | Arg203Ser | 39.0 ± 2.52 | 4.58 ± 0.26 | 118 ± 3.60 (67%) | 1.93 ± 0.15 | 4.72 ± 0.14 | 2.46 ± 0.21 (81%) |
| CYP2A6*17 | 5065G>A | Val365Met | 25.9 ± 6.16 | 1.70 ± 0.21 | 68.3 ± 12.2* (39%) | 1.72 ± 0.03 | 2.76 ± 0.08* | 1.60 ± 0.02** (53%) |
| CYP2A6*18 | 5668A>T | Tyr392Phe | 82.1 ± 7.42 | 7.94 ± 0.63 | 97.1 ± 6.86 (55%) | 2.77 ± 0.18 | 8.03 ± 0.43 | 2.90 ± 0.05 (95%) |
| CYP2A6*19 | 5668A>T | Tyr392Phe | 405 ± 118 | 2.29 ± 0.46 | 5.80 ± 0.53* (3%) | 4.75 ± 0.14*** | 2.18 ± 0.08* | 0.46 ± 0.01*** (15%) |
| 6558T>C | Ile471Thr | |||||||
| CYP2A6*21 | 6573A>G | Lys476Arg | 40.3 ± 3.69 | 6.15 ± 0.33 | 153 ± 9.35 (87%) | 2.34 ± 0.29 | 6.20 ± 0.35 | 2.67 ± 0.20 (88%) |
| CYP2A6*22 | 1794C>G | Asp158Glu | 53.8 ± 1.70 | 6.26 ± 0.36 | 116 ± 6.49 (66%) | 2.40 ± 0.10 | 5.28 ± 0.20 | 2.20 ± 0.06* (72%) |
| 1798C>A | Leu160Ile | |||||||
| CYP2A6*23 | 2161C>T | Arg203Cys | 41.1 ± 3.50 | 3.73 ± 0.32 | 91.1 ± 8.08* (52%) | 1.88 ± 0.04 | 4.86 ± 0.17 | 2.59 ± 0.04 (85%) |
| CYP2A6*24 | 594G>A | Val110Leu | 52.4 ± 8.56 | 4.79 ± 0.30 | 93.4 ± 12.1 (53%) | 2.22 ± 0.18 | 3.23 ± 0.11 | 1.46 ± 0.07*** (48%) |
| 6458A>T | Asn438Tyr | |||||||
| CYP2A6*25d | 1672T>C | Phe118Leu | 59.9 ± 5.94 | 1.67 ± 0.09 | 28.0 ± 2.48* (16%) | N.D. | N.D. | N.D. |
| CYP2A6*26a,c | 1672T>C | Phe118Leu | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. |
| 1703G>T | Arg128Leu | |||||||
| 1711T>G | Ser131Ala | |||||||
| CYP2A6*28 | 5745A>G | Asn418Asp | 35.0 ± 4.19 | 6.30 ± 0.73 | 181 ± 14.7 (103%) | 1.43 ± 0.12 | 5.05 ± 0.25 | 3.55 ± 0.23 (117%) |
| 5750G>C | Glu419Asp | |||||||
| CYP2A6*31 | 16A>C | Met6Leu | 35.4 ± 6.15 | 6.30 ± 0.30 | 184 ± 36.3 (105%) | 1.61 ± 0.07 | 5.64 ± 0.13 | 3.50 ± 0.12 (115%) |
| CYP2A6*35 | 6458A>T | Asn438Tyr | 75.7 ± 14.9 | 3.98 ± 0.51 | 53.5 ± 5.31* (30%) | 2.50 ± 0.28 | 4.63 ± 0.35 | 1.86 ± 0.08*** (61%) |
| CYP2A6*36b,d | 6458A>T | Asn438Tyr | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. |
| 6558T>C | Ile471Thr | |||||||
| CYP2A6*37b,c | 6458A>T | Asn438Tyr | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. |
| 6558T>C | Ile471Thr | |||||||
| 6600G>T | Arg485Leu | |||||||
| CYP2A6*38 | 5023T>C | Tyr351His | 108 ± 9.63* | 2.37 ± 0.12 | 22.2 ± 2.84* (13%) | 1.51 ± 0.08 | 2.07 ± 0.02* | 1.37 ± 0.07*** (45%) |
| CYP2A6*39 | 468G>A | Val68Met | 93.7 ± 20.4 | 1.91 ± 0.03 | 21.4 ± 4.72* (12%) | 1.50 ± 0.20 | 1.85 ± 0.02* | 1.26 ± 0.16* (41%) |
| CYP2A6*40 | 1767C>G | Ile149Met | 168 ± 72.0 | 1.97 ± 0.31 | 13.0 ± 2.93* (7%) | 1.34 ± 0.08* | 1.33 ± 0.08* | 0.99 ± 0.01** (33%) |
| CYP2A6*41 | 3515G>A | Arg265Gln | 263 ± 82.3 | 2.15 ± 0.43 | 8.43 ± 0.95* (5%) | 2.32 ± 0.23 | 1.26 ± 0.04* | 0.54 ± 0.04*** (18%) |
| CYP2A6*42 | 3524T>C | Ile268Thr | 450 ± 72.9 | 2.72 ± 0.37 | 6.07 ± 0.24* (3%) | 3.25 ± 0.44 | 0.63 ± 0.02* | 0.20 ± 0.02*** (6%) |
| CYP2A6*43 | 4406C>T | Thr303Ile | 1049 ± 683 | 4.76 ± 2.00 | 5.33 ± 1.28* (3%) | 8.55 ± 1.47 | 1.34 ± 0.09* | 0.16 ± 0.02*** (5%) |
| CYP2A6*44d | 5661G>A | Glu390Lys | 1097 ± 284 | 2.39 ± 0.43 | 2.22 ± 0.16* (1%) | N.D. | N.D. | N.D. |
| 5745A>G | Asn418Asp | |||||||
| 5750G>C | Glu419Asp | |||||||
| CYP2A6*45 | 6531C>T | Leu462Pro | 55.1 ± 11.3 | 1.19 ± 0.12 | 22.0 ± 2.24* (13%) | 1.65 ± 0.16 | 1.07 ± 0.02* | 0.65 ± 0.05*** (21%) |
N.D., Not determined.
* P < 0.05, **P < 0.01, and ***P < 0.005 compared with CYP2A6.1.
↵a The kinetic parameters for nicotine C-oxidation of CYP2A6.2, CYP2A6.5, CYP2A6.6, and CYP2A6.26 could not be determined because the enzymatic activity of P450 was not detected at the highest substrate concentration (1000 μas not detec
↵b The kinetic parameters for nicotine C-oxidation of CYP2A6.10, CYP2A6.36, and CYP2A6.37 could not be determined because the amount of product, cotinine, produced by these variants was below the quantification limit at low substrate concentrations.
↵c The kinetic parameters of CYP2A6.2, CYP2A6.5, CYP2A6.6, CYP2A6.26, and CYP2A6.37 could not be determined because the enzymatic activity of P450 was not detected at the highest substrate concentration (25 μas not detec
↵d The kinetic parameters for nicotine C-oxidation of CYP2A6.10, CYP2A6.25, CYP2A6.36, and CYP2A6.44 could not be determined because the amount of product, 7-hydroxycoumarin, produced by these variants was below the quantification limit at low substrate concentrations.