Increased Phenacetin Oxidation upon the L382V Substitution in Cytochrome P450 1A2 is Associated with Altered Substrate Binding Orientation
Abstract
:1. Introduction
2. Results
2.1. Purification of CYP1A2 Enzymes
2.2. Interactions of Phenacetin with CYP1A2 Enzymes
2.3. Determination of Spin State in CYP1A2 Enzymes
2.4. Nuclear Magnetic Resonance (NMR) T1 Experiments
2.5. Position of Phenacetin Relative to Heme in CYP1A2 Wild Type (WT) and Mutants
3. Discussion
4. Materials and Methods
4.1. Materials
4.2. Protein Expression and Purification
4.3. Binding Constant Determination
4.4. Spin State Determination
4.5. Enzyme Preparation for NMR
4.6. NMR Spectroscopy
4.7. Proton-Heme Iron Distance Calculations
4.8. Molecular Modeling: General
4.9. Substrate Docking with Distance Restraints
Author Contributions
Acknowledgments
Conflicts of Interest
Abbreviations
CYP | Cytochrome P450 |
WT | Wild Type |
EDTA | Ethlenediaminetetraacetic Acid |
1H NMR | Proton Nuclear Magnetic Resonance |
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Enzyme | Spectrum Type () | Ks |
---|---|---|
(nm) | (μM) | |
CYP1A2 WT | I (390–420) | 17.1 ± 0.6 |
CYP1A2 N312L | I (390–420) | 10.2 ± 0.4 |
CYP1A2 L382V | I (390–420) | 0.7 ± 0.1 |
CYP1A2 L382V/N312L | I (390–420) | 3.5 ± 0.2 |
Enzyme (+Substrate) | Low Spin | High Spin |
---|---|---|
% | ||
CYP1A2 WT (no substrate) | 90 | 10 |
+Phenacetin | 73 | 27 |
CYP1A2 N312L (no substrate) | 94 | 6 |
+Phenacetin | 86 | 14 |
CYP1A2 L382V (no substrate) | 95 | 5 |
+Phenacetin | 79 | 21 |
CYP1A2 L382V/N312L (no substrate) | 94 | 6 |
+Phenacetin | 83 | 17 |
Proton a | CYP1A2 WT b | CYP1A2 L382V c | ||||||
---|---|---|---|---|---|---|---|---|
T1,Fe3+ | T1,Fe2+-CO | r f | R g | T1,Fe3+ | T1,Fe2+-CO | r f | R g | |
Å | Å | |||||||
2,6 | 2.22 (0.11) | 2.78 (0.05) | 6.76 (0.34) | 8.25 | 2.28 (0.11) | 3.06 (0.04) | 6.34 (0.31) | 7.96 |
3.5 | 2.02 (0.04) | 2.50 (0.01) | 6.72 (0.43) | 8.32 | 2.39 (0.22) | 3.21 (0.08) | 6.39 (0.28) | 7.87 |
–OCH2– | 1.54 (0.15) | 1.85 (0.18) | 6.55 (0.87) | 8.01 | 2.43 (0.18) | 4.10 (0.07) | 5.93 (0.31) | 6.95 |
–COCH3 | 1.53 (0.07) | 1.62 (0.06) | 7.85 (0.81) | 9.44 | 1.64 (0.13) | 1.87 (0.11) | 6.79 (0.71) | 8.05 |
–CH3 | 1.33 (0.07) | 1.53 (0.06) | 6.69 (0.45) | 8.64 | 2.03 (0.13) | 2.82 (0.11) | 6.13 (0.89) | 7.58 |
Proton a | CYP1A2 N312L d | CYP1A2 L382V/N312Le | ||||||
T1,Fe3+ | T1,Fe2+-CO | R f (Å) | R g(Å) | T1,Fe3+ | T1,Fe2+-CO | r f (Å) | R g (Å) | |
Å | Å | |||||||
2,6 | 2.32 (0.10) | 2.45 (0.10) | 8.03 (0.42) | 9.11 | 2.43 (0.07) | 3.00 (0.06) | 6.72 (0.31) | 7.28 |
3.5 | 1.94 (0.09) | 2.04 (0.10) | 7.94 (0.43) | 9.38 | 2.95 (0.09) | 3.70 (0.11) | 6.87 (0.47) | 7.25 |
–OCH2– | 1.62 (0.04) | 1.69 (0.04) | 7.84 (0.77) | 8.67 | 1.98 (0.12) | 2.90 (0.09) | 5.96 (0.35) | 7.24 |
–COCH3 | 1.54 (0.12) | 1.60 (0.11) | 8.19 (1.12) | 9.34 | 2.12 (0.14) | 2.59 (0.12) | 6.62 (0.15) | 8.01 |
–CH3 | 1.87 (0.12) | 1.97 (0.07) | 7.83 (0.52) | 8.94 | 2.23 (0.09) | 2.61 (0.11) | 6.92 (0.34) | 7.47 |
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Huang, Q.; Szklarz, G.D. Increased Phenacetin Oxidation upon the L382V Substitution in Cytochrome P450 1A2 is Associated with Altered Substrate Binding Orientation. Int. J. Mol. Sci. 2018, 19, 1580. https://doi.org/10.3390/ijms19061580
Huang Q, Szklarz GD. Increased Phenacetin Oxidation upon the L382V Substitution in Cytochrome P450 1A2 is Associated with Altered Substrate Binding Orientation. International Journal of Molecular Sciences. 2018; 19(6):1580. https://doi.org/10.3390/ijms19061580
Chicago/Turabian StyleHuang, Qingbiao, and Grazyna D. Szklarz. 2018. "Increased Phenacetin Oxidation upon the L382V Substitution in Cytochrome P450 1A2 is Associated with Altered Substrate Binding Orientation" International Journal of Molecular Sciences 19, no. 6: 1580. https://doi.org/10.3390/ijms19061580