Toxicological Research

Korean Society of Toxicology & Korea Environmental Mutagen Society (한국독성학회)
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Catalytic enhancements in cytochrome P450 2C19 by cytochrome b5

  • Gyu-Hyeong Lee (Department of Biological Sciences, Konkuk University) ;
  • Vitchan Kim (Department of Biological Sciences, Konkuk University) ;
  • Sung-Gyu Lee (Department of Biological Sciences, Konkuk University) ;
  • Eunseo Jeong (Department of Biological Sciences, Konkuk University) ;
  • Changmin Kim (Department of Biological Sciences, Konkuk University) ;
  • Yoo-Bin Lee (Department of Biological Sciences, Konkuk University) ;
  • Donghak Kim (Department of Biological Sciences, Konkuk University)
  • Received : 2023.08.08
  • Accepted : 2023.11.23
  • Published : 2024.04.15
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Abstract

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 Escherichia coli 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 Kd 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 b5 stimulated a five-times catalytic turnover number of P450 2C19 with a kcat 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 b5 affects the metabolism of P450 2C19 to drugs and steroids.

Keywords

Acknowledgement

This paper was supported by an NRF grant funded by the Korean government (NRF-2019R1A2C1004722) and Konkuk University Researcher Fund in 2022.

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