Biomol Ther  https://doi.org/10.4062/biomolther.2019.112
Functional Characterization of Pharmcogenetic Variants of Human Cytochrome P450 2C9 in Korean Populations
Myung-A Cho1, Jihoon G. Yoon2, Vitchan Kim1, Harim Kim1, Rowoon Lee1, Min Goo Lee2 and Donghak Kim1,*
1Department of Biological Sciences, Konkuk University, Seoul 05029,2Department of Pharmacology, Yonsei University College of Medicine, Seoul 03722, Republic of Korea
E-mail: donghak@konkuk.ac.kr
Tel: +82-2-450-3366, Fax: +82-2-3436-5432
Received: July 1, 2019; Revised: July 31, 2019; Accepted: August 1, 2019; Published online: September 5, 2019.
© The Korean Society of Applied Pharmacology. All rights reserved.

Abstract
Human cytochrome P450 2C9 is a highly polymorphic enzyme that is required for drug and xenobiotic metabolism. Here, we studied eleven P450 2C9 genetic variants—including three novel variants F69S, L310V, and Q324X—that were clinically identified in Korean patients. P450 2C9 variant enzymes were expressed in Escherichia coli and their bicistronic membrane fractions were prepared The CO-binding spectra were obtained for nine enzyme variants, indicating P450 holoenzymes, but not for the M02 (L90P) variant. The M11 (Q324X) variant could not be expressed due to an early nonsense mutation. LC-MS/MS analysis was performed to measure the catalytic activities of the P450 2C9 variants, using diclofenac as a substrate. Steady-state kinetic analysis revealed that the catalytic efficiency of all nine P450 2C9 variants was lower than that of the wild type P450 2C9 enzyme. The M05 (R150L) and M06 (P279T) variants showed high kcat values; however, their Km values were also high. As the M01 (F69S), M03 (R124Q), M04 (R125H), M08 (I359L), M09 (I359T), and M10 (A477T) variants exhibited higher Km and lower kcat values than that of the wild type enzyme, their catalytic efficiency decreased by approximately 50-fold compared to the wild type enzyme. Furthermore, the novel variant M07 (L310V) showed lower kcat and Km values than the wild type enzyme, which resulted in its decreased (80%) catalytic efficiency. The X-ray crystal structure of P450 2C9 revealed the presence of mutations in the residues surrounding the substrate-binding cavity. Functional characterization of these genetic variants can help understand the pharmacogenetic outcomes.
Keywords: Cytochrome P450, P450 2C9, Diclofenac, Polymorphism, Pharmacogenetics

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