biostudies-literatureUnknown40(2)Lee GHHuman cytochrome P450 2C19 catalyzes P450 enzyme reactions of various substrates, including steroids and clinical drugs. Recombinant P450 2C19 enzyme with histidine tag was successfully expressed in <i>Escherichia coli</i> and purified using affinity column chromatography. Ultra-performance liquid chromatography-tandem mass (UPLC-MS/MS) spectrometry showed that the purified P450 2C19 enzyme catalyzed 5-hydroxylation reaction of omeprazole. The purified enzyme displayed typical type I binding spectra to progesterone with a <i>K</i>_d value of 4.5 ± 0.2 µM, indicating a tight substrate binding. P450 2C19 catalyzed the hydroxylation of progesterone to produce 21-hydroxy (OH) as a major and 17-OH product as a minor product. Steady-state kinetic analysis of progesterone 21-hydroxylation indicated that the addition of cytochrome <i>b</i>₅ stimulated a five-times catalytic turnover number of P450 2C19 with a <i>k</i>_cat value of 1.07 ± 0.08 min^-1. The molecular docking model of progesterone in the active site of P450 2C19 displayed that the 21-carbon of progesterone was located close to the heme with a distance of 4.7 Å, suggesting 21-hydroxylation of progesterone is the optimal reaction of P450 2C19 enzyme for a productive orientation of the substrate. Our findings will help investigate the extent to which cytochrome <i>b</i>₅ affects the metabolism of P450 2C19 to drugs and steroids.<h4>Supplementary information</h4>The online version contains supplementary material available at 10.1007/s43188-023-00219-8.Toxicological research215-222https://www.ebi.ac.uk/biostudies/studies/S-EPMC10959859biostudies-literatureCatalytic enhancements in cytochrome P450 2C19 by cytochrome &lt;i&gt;b&lt;/i&gt;&lt;sub&gt;5&lt;/sub&gt;.PMC10959859Kim CKim DLee YBJeong ELee SGKim VLee GHfalseCatalytic enhancements in cytochrome P450 2C19 by cytochrome &lt;i&gt;b&lt;/i&gt;&lt;sub&gt;5&lt;/sub&gt;.Human cytochrome P450 2C19 catalyzes P450 enzyme reactions of various substrates, including steroids and clinical drugs. Recombinant P450 2C19 enzyme with histidine tag was successfully expressed in <i>Escherichia coli</i> and purified using affinity column chromatography. Ultra-performance liquid chromatography-tandem mass (UPLC-MS/MS) spectrometry showed that the purified P450 2C19 enzyme catalyzed 5-hydroxylation reaction of omeprazole. The purified enzyme displayed typical type I binding spectra to progesterone with a <i>K</i>_d value of 4.5 ± 0.2 µM, indicating a tight substrate binding. P450 2C19 catalyzed the hydroxylation of progesterone to produce 21-hydroxy (OH) as a major and 17-OH product as a minor product. Steady-state kinetic analysis of progesterone 21-hydroxylation indicated that the addition of cytochrome <i>b</i>₅ stimulated a five-times catalytic turnover number of P450 2C19 with a <i>k</i>_cat value of 1.07 ± 0.08 min^-1. The molecular docking model of progesterone in the active site of P450 2C19 displayed that the 21-carbon of progesterone was located close to the heme with a distance of 4.7 Å, suggesting 21-hydroxylation of progesterone is the optimal reaction of P450 2C19 enzyme for a productive orientation of the substrate. Our findings will help investigate the extent to which cytochrome <i>b</i>₅ affects the metabolism of P450 2C19 to drugs and steroids.<h4>Supplementary information</h4>The online version contains supplementary material available at 10.1007/s43188-023-00219-8.2024-01-01T00:00:00Z2024 Apr2026-06-01T10:13:40.699Z2025-07-13T03:04:43.193ZS-EPMC109598593852513710.1007/s43188-023-00219-8