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394
Regular Article
Biol. Pharm. Bull. 35(3) 394 399 (2012)
Vol. 35, No. 3
Functional Characterization of Allelic Variants of Polymorphic Human
Cytochrome P450 2A6 (CYP2A6*5, *7, *8, *18, *19, and *35)
Songhee Han,a Seunghye Choi,a Young-Jin Chun,b Chul-Ho Yun,c Chang Hoon Lee,d
,a
Hee Jung Shin,e Han Sung Na,e Myeon Woo Chung,e and Donghak Kim*
a Department of Biological Sciences and Center for Biotechnology Research in UBITA (CBRU), Konkuk University;
b
c
Seoul 143–701, Korea: College of Pharmacy, Chung-Ang University; Seoul 156–756, Korea: School of Biological
d
Sciences and Technology, Chonnam National University; Gwangju 500–757, Korea: College of Pharmacy, Dongguk
University; Seoul 100–715, Korea: and National Institute of Food and Drug Safety Evaluation, Korea Food & Drug
e
Administration; Chungcheongbuk-do 363–951, Korea.
Received October 13, 2011; accepted December 13, 2011; published online December 22, 2011
Cytochrome P450 2A6 (CYP2A6) catalyzes important metabolic reactions of many xenobiotic com-
pounds, including coumarin, nicotine, cotinine, and clinical drugs. Genetic polymorphisms of CYP2A6 can
influence its metabolic activities. This study analyzed the functional activities of six CYP2A6 allelic vari-
ants (CYP2A6*5, *7, *8, *18, *19, and *35) containing nonsynonymous single-nucleotide polymorphisms.
Recombinant variant enzymes of CYP2A6*7, *8, *18, *19, and *35 were successfully expressed in Escherichia
coli and purified. However, a P450 holoenzyme spectrum was not detected for the CYP2A6*5 allelic vari-
ant (G479V). Structural analysis shows that the G479V mutation may alter the interaction between the A
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helix and the F G helices. Enzyme kinetic analyses indicated that the effects of mutations in CYP2A6 al-
lelic variants on drug metabolism are dependent on the substrates. In the case of coumarin 7-hydroxylation,
CYP2A6*8 and *35 displayed increased Km values whereas CYP2A6*18 and *19 showed decreased kcat values,
which resulted in lower catalytic efficiencies (kcat/Km). In the case of nicotine 5-oxidation, the CYP2A6*19
variant exhibited an increased Km value, whereas CYP2A6*18 and *35 showed much greater decreases in kcat
values. These results suggest that individuals carrying these allelic variants are likely to have different me-
tabolisms for different CYP2A6 substrates. Functional characterization of these allelic variants of CYP2A6
can help determine the importance of CYP2A6 polymorphisms in the metabolism of many clinical drugs.
Key words P450; CYP 2A6; allelic variant; polymorphism; coumarin; nicotine
The cytochrome P450 (CYP, P450) family is composed of CYP2A6. The substrate preferences of these mutant enzymes
major phase I enzymes responsible for metabolizing xenobi- are different from those of the wild-type enzymes, and there-
otic chemicals including clinical drugs.1) CYP2A6 was first fore, the mutant enzymes can produce dramatic effects in
determined to catalyze coumarin 7-hydroxylation in the hu- CYP2A6 metabolism.
man liver.2) CYP2A6 also metabolizes many clinical drugs,
In this study, we analyzed the functional activities of six
including valproic acid, losigamone, chlormethiazole, letro- allelic variants of CYP2A6 (CYP2A6*5, *7, *8, *18, *19, and
zole, fadrozole, methoxyflurane, and tegafur.3) This enzyme *35) containing nonsynonymous SNPs. They contain substi-
is known to catalyze the metabolism of tobacco-specific com- tutions of the following amino acids: CYP2A6*5 (G479V);
pounds such as nicotine, cotinine, 4-(methylnitrosamino)-1-(3- CYP2A6*7 (I471T); CYP2A6*8 (R485L); *18 (Y392F; *19
the bioactivation of procarcinogens has also been intensively ki.se/). These variants are frequently found in Asian popula-
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8)
investigated.4
tions. The allelic frequencies of CYP2A6*5, *7, *8, *18, and
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Genetic variations in metabolic enzymes can cause dra- *35 in Japanese and Koreans were 0.5%, 9.8%, 1.4 2.2%,
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13 19)
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matic differences in the response to specific drugs. Some 0.5%, and 0.5 0.8%, respectively.
In view of the impor-
drug-metabolizing P450 enzymes are very polymorphic, and tance of CYP2A6 in the metabolism of many clinical drugs
the study of their polymorphisms is of particular interest be- and several tobacco-specific compounds, we determined the
cause it can help develop a rational means to optimize drug functional changes in these allelic variants.
therapy and ensure maximum efficacy with minimal adverse
effects. Previous studies reported the genetic polymorphisms
of CYP2A6 and proposed their relevance to cancer risk due
MATERIALS AND METHODS
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12)
to variations in nicotine and N-nitrosoamine metabolism.9
Chemicals and Enzymes Coumarin, 7-hydroxycoumarin,
Interestingly, the ethnic distribution of CYP2A6 polymor- nicotine, imidazole, 3-[(3-cholamidopropyl)dimethylammonio]-
phisms is slightly different from that of other P450 enzymes. 1-propanesulfonate (CHAPS), dilauroyl-L-phosphatidylcholine
A very low frequency of poor metabolizers (PMs) has been (DLPC), NADP+, and NADPH were purchased from Sigma
reported in Caucasians and Africans, but the occurrence of Chemical (St. Louis, MO, U.S.A.) or Aldrich Chemical Co.
PMs is much more common in Asians.3) To date, at least 36 (Milwaukee, WI, U.S.A.). Ni2+-Nitrilotriacetate agarose
allelic variants of CYP2A6 have been identified, 26 of which was purchased from Qiagen (Valencia, CA, U.S.A.). Other
are located in the coding region of the CYP2A6 gene (http:// chemicals were of the highest commercially available grade.
www.cypalleles.ki.se/). Nonsynonymous single nucleotide Escherichia coli DH5α cells were purchased from Invitrogen
polymorphisms (SNPs) can alter the protein sequences of (Carlsbad, CA, U.S.A.). Rat NADPH-P450 reductase was het-
*To whom correspondence should be addressed. e-mail: donghak@konkuk.ac.kr
© 2012 The Pharmaceutical Society of Japan