Catalytic enhancements in cytochrome P450 2C19 by cytochrome b 5

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 and purified using affinity column chromatography. Ultra-performance liquid chromatography-tandem mas...

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Veröffentlicht in:Toxicological research (Seoul) 2024-04, Vol.40 (2), p.215
Hauptverfasser: Lee, Gyu-Hyeong, Kim, Vitchan, Lee, Sung-Gyu, Jeong, Eunseo, Kim, Changmin, Lee, Yoo-Bin, Kim, Donghak
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Sprache:eng
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Zusammenfassung: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 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 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 stimulated a five-times catalytic turnover number of P450 2C19 with a value of 1.07 ± 0.08 min . 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 affects the metabolism of P450 2C19 to drugs and steroids. The online version contains supplementary material available at 10.1007/s43188-023-00219-8.
ISSN:1976-8257
DOI:10.1007/s43188-023-00219-8