Title Page
Contents
ABSTRACT 9
1. Introduction 11
1.1. Cytochrome P450 (CYP) 11
1.2. Human cytochrome P450 12
1.3. Cytochrome P450 2C19 14
1.4. Cytochrome b₅ 16
2. Materials and Methods 18
2.1. Materials 18
2.2. Construction of P450 2C19 with His-tag Expression Plasmids 19
2.3. Expression and Purification of Recombinant P450 2C19 Enzyme 21
2.4. Progesterone Binding Titration Analysis 22
2.5. Omeprazole 5-Hydroxylation Assay 23
2.6. Progesterone 21-Hydroxylation Assay 24
2.7. Molecular Docking Modeling of P450 2C19 25
3. RESULTS 26
3.1. Expression and Purification of P450 2C19 26
3.2. Binding Analysis of P450 2C19 to Progesterone 30
3.3. Omeprazole 5-Hydroxylation and Stimulation by Cytochrome b₅ 32
3.4. Progesterone Hydroxylation and Stimulation by Cytochrome b₅ 37
3.5. Docking Model of P450 2C19 with Progesterone 41
4. DISCUSSION 44
References 48
Abstract (in Korean) 52
Table 1. Classification of human cytochrome P450s based on major substrate class. 13
Table 2. Steady-state kinetic parameters for omeprazole 5-hydroxylation by P450 2C19 and stimulation effect by b₅. 36
Table 3. Steady-state kinetic parameters for progesterone 21-hydroxylation by P450 2C19 and stimulation effect by b₅. 40
Figure 1. Oxidation reactions by P450 2C19. 15
Figure 2. Cytochrome P450 17A1 with cytochrome b₅ and NPR (CPR) in a lipid bilayer. 17
Figure 3. Schematic illustration of cloning of CYP2C19. 20
Figure 4. Expression of human CYP2C19 wild-type. 27
Figure 5. Purification of human CYP2C19 wild type. 28
Figure 6. Absolute spectra of P450 2C19 enzyme. 29
Figure 7. Binding analysis of P450 2C19 to progesterone. 31
Figure 8. UPLC-mass spectrometry chromatograms of omeprazole and 5-hydroxyomeprazole. 33
Figure 9. Omeprazole hydroxylation by P450 2C19 and stimulatory effect of b₅. 34
Figure 10. Steady state kinetic analysis of omeprazole 5-hydroxylation by P450 2C19 and stimulatory effect of b₅. 35
Figure 11. UPLC-mass spectrometry chromatograms of progesterone and 21-hydroxyprogesterone. 38
Figure 12. Steady-state kinetic analysis of progesterone hydroxylation by P450 2C19 and stimulatory effect of b₅. 39
Figure 13. Molecular docking modeling of P450 2C19 with progesterone. 42
Figure 14. Molecular docking modeling of P450 2C19 with progesterone. 43