CYP2A13-catalysed coumarin metabolism: Comparison with CYP2A5 and CYP2A6

Article abstract of DOI:10.1080/0049825021000022302

1. We investigated the total metabolism of coumarin by baculovirus (BV)-expressed CYP2A13 and compared it with metabolism by BV-expressed CYP2A6. The major coumarin metabolite formed by CYP2A13 was 7-hydroxycoumarin, which accounted for 43% of the total metabolism. The product of 3,4-epoxidation, o-hydroxyphenylacetaldehyde (o-HPA), accounted for 30% of the total metabolites. 2. The K_m and V_max for CYP2A13-mediated coumarin 7-hydroxylation were 0.48 ± 0.07 μM and 0.15 ± 0.006 nmol min^-1 nmol^-1 CYP, respectively. The V_max of coumarin 7-hydroxylation by CYP2A13 was about 16-fold lower than that of CYP2A6, whereas the K_m was 10-fold lower. 3. In the mouse, there were two orthologues for CYP2A6: CYP2A4 and CYP2A5, which differed by only 11 amino acids. However, CYP2A5 is an efficient coumarin 7-hydroxylase, where as CYP2A4 is not. We report here that BV-expressed CYP2A4 metabolizes coumarin by 3,4-epoxidation. Two products of the 3,4-epoxidation pathway, o-HPA and o-hydroxyphenylacetic acid (o-HPAA), were detected by radioflow HPLC. 4. The K_m and V_max for the coumarin 3,4-epoxidation by CYP2A4 were 8.7 ± 3.6 μM and 0.20 ± 0.04nmol min^-1 nmol^-1 CYP, respectively. Coumarin 7-hydroxylation by CYP2A5 was more than 200 times more efficient than 3,4 epoxidation by CYP2A4.

Full text of DOI:10.1080/0049825021000022302

xenobiotica, 2003, vol. 33, no. 1, 73–81
CYP2A13-catalysed coumarin metabolism:
comparison with CYP2A5 and CYP2A6
L. B. VON WEYMARN and S. E. MURPHY*
Department of Biochemistry, Molecular Biology & Biophysics, University of Minnesota
Cancer Center, Mayo Mail Code 806, 420 Delaware Street SE., Minneapolis, MN 55455,
USA
Received 8 May 2002
1. We investigated the total metabolism of coumarin by baculovirus (BV)-expressed
CYP2A13 and compared it with metabolism by BV-expressed CYP2A6. The major
coumarin metabolite formed by CYP2A13 was7-hydroxycoumarin, which accounted for
43% of the total metabolism. The product of 3,4-epoxidation, o-hydroxyphenylacetalde-
hyde (o-HPA), accounted for 30% of the total metabolites.
2. The Km and Vmax for CYP2A13-mediated coumarin 7-hydroxylation were
0.48 Æ 0.07 mm and 0.15 Æ 0.006 nmol minÀ1 nmolÀ1 CYP, respectively. The Vmax of
coumarin 7-hydroxylation by CYP2A13 wasabout 16-fold lower than that of CYP2A6,
whereasthe Km was10-fold lower.
3. In the mouse, there were two orthologues for CYP2A6: CYP2A4 and CYP2A5,
which differed by only 11 amino acids. However, CYP2A5 is an efficient coumarin 7-
hydroxylase, where as CYP2A4 is not. We report here that BV-expressed CYP2A4
metabolizescoumarin by 3,4-epoxidation. Two productsof the 3,4-epoxidation pathway,
o-HPA and o-hydroxyphenylacetic acid (o-HPAA), were detected by radioflow HPLC.
4. The Km and Vmax for the coumarin 3,4-epoxidation by CYP2A4 were 8.7 Æ 3.6 mm
and 0.20 Æ 0.04 nmol minÀ1 nmolÀ1 CYP, respectively. Coumarin 7-hydroxylation by
CYP2A5 wasmore than 200 timesmore efficient than 3,4 epoxidation by CYP2A4.
Introduction
CYP2A13 isone of three membersof the human CYP2A gene family
(Fernandez-Salguero et al. 1995). The other membersare CYP2A6, a hepatic
coumarin 7-hydroxylase, and CYP2A7, reported to be a non-functional enzyme
(Yamano et al. 1990). A number of regions in the CYP2A13 sequence are similar to
CYP2A7, therefore CYP2A13 wasalso predicted to be non-functional (Ding et al.
1995). However, Su et al. (2000) recently expressed CYP2A13 in Spodoptera
frugiperda (Sf9) insect cells and reported that it is in fact a functional enzyme that
metabolizes several compounds which are substrates for CYP2A6. The coumarin
7-hydroxylase activity by CYP2A13 was previously measured at a single coumarin
concentration. At 100 mm NNK, CYP2A13 catalysed the 7-hydroxylation of
coumarin at about 10% of the rate of CYP2A6 (Su et al. 2000).
The highest level of CYP2A13 expression was found in the nasal mucosa;
however, 2A13 is also expressed in significant levels in human lung and trachea (Su
et al. 2000). CYP2A6 isprimarily hepatic, constituting 1–10% of total CYP in the
human liver (Yun et al. 1991, Shimada et al. 1994). CYP2A6 hasbeen detected in
small amounts in nasal mucosa and there are conflicting results about whether or
not CYP2A6 ispresent in the human lung (Crawford et al. 1998, Koskela et al.
* Author for correspondence; e-mail: murph062@umn.edu
Xenobiotica ISSN 0049–8254 print/ISSN 1366–5928 online # 2003 Taylor & FrancisLtd
http://www.tandf.co.uk/journals
DOI: 10.1080/0049825021000022302

74
L. B. von Weymarn and S. E. Murphy
1999, Su et al. 2000). Koskela et al. did not detect any CYP2A6 transcripts in
human lung using RT-PCR. In contrast, Crawford et al. detected CYP2A6
mRNA in human bronchial epithelial cells. The discrepancy observed might be
due to the detection of CYP2A13 with the primersused by Crawford et al. The
recognition site for these primers in CYP2A13 differs from 2A6 by only one
nucleotide. Using sequence-specific primers and competitive RT-PCR, Su et al.
(2000) reported that the amount of CYP2A13 mRNA in the lung iseight times
higher than that of CYP2A6.
CYP2A6 metabolizesthe carcinogens4-(methylnitrosamino)-1-(3-pyridyl)-1-
butanone (NNK) and N-nitrosonornicotine (NNN), which are tobacco-specific
nitrosamines (Patten et al. 1996, 1997). NNK isa potent lung carcinogen in
laboratory animalsand a likely human lung carcinogen (Hecht 1998). NNN
primarily inducesoesophageal tumoursin animalsand isbelieved to play a role
in tobacco-induced oral and oesophageal cancer in humans. The metabolism of
NNN by CYP2A6 isboth psecific and efficient (Patten et al. 1997); however,
CYP2A6-catalysed NNK metabolism is much less efficient (Patten et al. 1996).
CYP2A13 catalyses the activation of NNK by ꢀ-hydroxylation at a significantly
higher rate than does2A6 (Su et al. 2000). The Vmax/Km for NNK methylene
hydroxylation by 2A6 is0.008 compared with 0.36 for 2A13 (Patten et al. 1996, Su
et al. 2000). Since CYP2A13 ispresent in human lung, it islikely to catalyse NNK
activation in this tissue and therefore play an important role in tobacco related lung
cancers.
In the mouse there are two orthologues of CYP2A6: CYP2A4 and 2A5
(Honkakoski and Negishi 1997). These two enzymes are 98% homologous, differ-
ing by only 11 amino acids; however, they catalyse NNK metabolism with
strikingly different efficiencies (Felicia et al. 2000). CYP2A4 catalyses NNK ꢀ-
hydroxylation with a 54-fold lower efficiency (Vmax/Km) than doesCYP2A5.
However, the rate of CYP2A5-catalysed NNK ꢀ-hydroxylation is1000-fold
greater than that of CYP2A6 at 1 mm NNK (Felicia et al. 2000).
CYP2A5 and 2A6 are well-characterized coumarin 7-hydroxylases. However,
CYP2A4 hasbeen reported to have little or no coumarin 7-hydroxylase activity
(Lindberg and Negishi 1989, Negishi et al. 1989, Felicia et al. 2000). Coumarin 7-
hydroxylation is one of several pathways of coumarin metabolism (figure 1). In all
species studied to date, the predominant pathway is either 7-hydroxylation or the
formation of the ring-opened products o-hydroxyphenylacetaldehyde (o-HPA) and
o-hydroxyphenylacetic acid (o-HPAA) (Shilling et al. 1969, Egan and O’Kennedy
1992, Fentem and Fry 1993). o-HPA isgenerated non-enzymatically from the
unstable coumarin 3,4-epoxide, whereas o-HPAA isan oxidation product of o-
HPA (figure 1) (Born et al. 1997, 2000). 7-Hydroxylation isthe predominant
coumarin metabolism pathway in humans, due to the presence of CYP2A6 in the
liver (Miles et al. 1990). However, other human CYPsmetabolize coumarin by
pathwaysother than 7-hydroxylation. o-HPA isthe major metabolite formed by
CYP1A1, 1A2, 2B6, 2E1 and 3A4, although the rate of metabolism is quite low
(Zhuo et al. 1999).
In the current study, the coumarin 7-hydroxylase activity of CYP2A13 was
compared with that of CYP2A6. The kinetic parametersfor CYP2A13-mediated
coumarin 7-hydroxylation were determined and other CYP2A13-catalysed path-
ways of coumarin metabolism were quantified. Total coumarin metabolism by the
mouse CYP, 2A4, was also investigated and compared with the metabolism of

Coumarin metabolism by CYP2A enzymes
75
Figure 1. Pathwaysof coumarin metabolism.
coumarin by the highly homologousCYP, 2A5, aswell asthe human CYPs, 2A6
and 2A13.
Materials and methods
Chemicals
[U-14C-benzyl] coumarin (58 mCi mmolÀ1), purity >99.5% by HPLC analysis, was purchased from
Amersham Pharmacia Biotech (Piscataway, NJ, USA). 4-Hydroxycoumarin, 2-hydroxyphenylacetic
acid, o-coumaric acid and 6,7-dihydroxycoumarin were purchased from Fluka Chemical Co. (Ronkon-
koma, NY, USA). 3-Hydroxycoumarin wasobtained from Indofine Chemical Co. (Sommerville, NJ,
USA). o-Hydroxyphenylacetaldehyde wasa gift from Dr LoisD. Lehman-McKeeman of Procter &
Gamble (Cincinnati, OH, USA) and 5-hydroxycoumarin wasa gift from Dr Takahsi Harayama
(Okayama University, Japan). All other chemicals were purchased from Sigma Chemical Co. (St
Louis, MO, USA) or Aldrich Chemical Co. (Milwaukee, WI, USA).
Metabolismof 14C-coumarin
Homogenate of baculovirus (BV)-expressed CYP2A13 (Su et al. 2000) wasprovided by Dr Xinxin
Ding (Wadsworth Center, Albany, NY, USA). CYP2A4, CYP2A5 and CYP2A6 were expressed in Sf9
cells using the BV-expression system (Felicia et al. 2000), and microsomes were prepared as described.
Microsomes from BV-expressed CYP2A4 (33.9 pmol), CYP2A5 (1.8 pmol), CYP2A6 (1.8 pmol) and
CYP2A13 cell homogenate (7.2 pmol) were incubated with BV-expressed human P450:NADPH
oxidoreductase, 5 mm [U-14C-benzyl]-coumarin, and an NADPH-generating system (0.4 mm
NADP+, 100 mm glucose 6-phosphate, and 0.4 units mlÀ1 glucose 6-phosphate dehydrogenase) in a
200 ml total volume of 50 mm Trisbuffer (pH 7.4) for 30 min at 37 8C. A ratio of 1:5, CYP to

76
L. B. von Weymarn and S. E. Murphy
oxidoreductase, was used in all experiments. All analyses were performed in duplicate. The reactions
were terminated by the addition of 20 ml 15% trichloroacetic acid and then centrifuged. The supernatant
was mixed with standards and analysed by reverse-phase HPLC with radioflow detection. The
standards added were o-HPA, o-HPAA, o-hydroxyphenypropionic acid, o-coumaric acid, 5-, 6-, 7-,
and 8-hydroxycoumarin, coumarin, and 6,7-dihydroxycoumarin. A Phenomenex (Torrance, CA, USA)
Bondclone C18 column (3.9 Â 300 mm, 10 mm) was used for the analysis. The mobile phase consisted of
1% acetic acid in water (A) and 1% acetic acid in methanol (B). The metaboliteswere eluted isocratically
with 29% B and 71% A. The flow rate was1 ml min À1. The metabolism of coumarin was linear with time
and protein for all CYP enzymes. For the determination of kinetic parameters, CYP2A4 (18.4 pmol) was
incubated with 0.5–20 mm 14C-coumarin asdescribed above.
Coumarin 7-hydroxylation by CYP2A13
CYP2A13 (4.15 pmol) wasincubated with 0.1–20 mm coumarin (non-radioactive) for 10 min at 378C
as described above. The 7-hydroxycoumarin formed was analysed by HPLC with fluorescence
detection (excitation wavelength 350 nm, emission wavelength 453 nm) as described by von Weymarn
et al. (1999). Quantification was obtained by comparison to a standard curve of 7-hydroxycoumarin
versus peak area.
Kinetics and statistical analysi
Km and Vmax for coumarin metabolism by CYP2A13 and 2A4 were determined using the EZ-Fit 5
kinetics program from Perrella Scientific (Amherst, NH, USA). It uses a non-linear regression method
of curve fitting and the Runs test of residuals to determine statistically whether experimental data are
randomly distributed around the curve with 95% confidence (Perrella 1988).
Results and discussion
The metabolism of 14C-coumarin by CYP2A4, CYP2A5, CYP2A6 and
CYP2A13 was analysed by reverse-phase HPLC with radioflow detection (figures
2 and 4). The use of coumarin labelled with 14C in the benzene ring allowsthe
detection of other metabolites, aswell as7-hydroxycoumarin, and we previously
developed an HPLC system to separate most coumarin metabolites (von Weymarn
and Murphy 2001). The major metabolite of CYP2A6-catalysed coumarin meta-
bolism eluted at 20.3 min (figure 2A), namely 7-hydroxycoumarin. As expected,
thisisthe only metabolite detected (table 1) (Miles et al. 1990, Yamano et al.
1990). However, in contrast to CYP2A6, several radioactive metabolites of
CYP2A13-catalysed coumarin metabolism were detected (figure 2B). The major
metabolite was7-hydroxycoumarin (20.3 min), accounting for 43% of the total
metabolism (table 1). However, a product of 3,4-epoxidation, o-HPA, (14.2 min),
was also formed at a significant rate accounting for 30% of the total metabolism.
Two hydroxylated coumarins, 6-hydroxy (6-OH, 15.6 min) and 8-hydroxycou-
marin (8-OH, 16.4 min), were also detected. The formation of all of these products
was linear with enzyme and time under the conditions studied (data not shown).
The early eluting peak (5 min) was present in control samples and dependent on
the presence of Sf9 microsomes (figure 2C). The metabolism of coumarin by
CYP2A13 is clearly not as specific as metabolism by CYP2A6 despite the 94%
sequence homology between the two enzymes.
The kinetic parametersfor 7-hydroxycoumarin formation by CYP2A13 were
determined using reverse-phase HPLC with fluorescence detection. The forma-
tion of 7-hydroxycoumarin followed simple Michaelis–Menten kinetics (figure 3).
The Km for coumarin 7-hydroxylation was0.48 Æ 0.07 mm, the Vmax
0.15 Æ 0.006 nmol minÀ1 nmolÀ1 CYP. This Km is similar to that of BV-expressed
CYP2A5 (1.2 mm; Felicia et al. 2000) and about 10-fold lower than that of BV-
expressed CYP2A6 (6 mm; Zhuo et al. 1999). The Vmax for CYP2A13-catalysed

Coumarin metabolism by CYP2A enzymes
77
Figure 2. Radioflow HPLC analysis of coumarin metabolites. 14C-coumarin (5 mm wasincubated for
30 min with 1.8 pmol CYP2A6 (A), 7.2 pmol CYP2A13 (B) or without enzyme (C) asdescribed
in the Materials and methods. The products of the reaction were co-injected with standards on a
C18 HPLC column.
Table 1. Relative metabolism of coumarin by two major pathways.a
b
Metabolites(% of total)
Mouse
Human
CYP2A4
CYP2A5
CYP2A6
CYP2A13
7-Hydroxylation
3,4-Epoxidationc
0
98
79
7
100
0
43
30
a CYP2A4 (33.9 pmol), CYP2A5 (1.8 pmol), CYP2A6 (1.8 pmol) or CYP2A13 (7.2 pmol) were
incubated for 30 min with 5 mm coumarin as described in the Materials and methods. The products were
analysed by radioflow HPLC.
b Averagesof duplicate determinations.
c Valuesdetermined for the 3,4-epoxide pathway are the sum of o-HPA and o-HPAA.

78
L. B. von Weymarn and S. E. Murphy
Figure 3. Kinetic analysis of 7-hydroxycoumarin formation by CYP2A13. Coumarin (0.5–10 mm) was
incubated for 10 min with 4.15 pmol CYP2A13 as described in the Materials and methods. The
formation of 7-hydroxycoumarin was determined by HPLC analysis with fluorescence detection.
Curves were graphed by non-linear regression kinetic parameters determined using EZ-Fit 5.
The insert is a Lineweaver–Burk plot of the kinetic analysis. The data points are the average of
two sets of duplicate determinations.
coumarin 7-hydroxylation isabout 16-fold lessthan that of CYP2A5 and CYP2A6
(Zhuo et al. 1999, Felicia et al. 2000).
Coumarin metabolism by CYP2A4 and 2A5, two mouse orthologues of
CYP2A6, was next investigated using 14C-coumarin (figure 4). The major meta-
bolitesof CYP2A4-mediated coumarin metabolims were
o-HPA (13.5 min,
figure 4B) and o-HPAA (12.1 min), productsof the coumarin 3,4-epoxide path-
way. 7-Hydroxycoumarin wasnot detected (limit of detection <0.1 pmol) (figure
4A and table 1). The formation of productsfrom the 3,4-epoxide pathway
accounted for 98% of the coumarin metabolism by CYP2A4 (table 1). An unknown
metabolite, eluting at 7.5 min, accounted for the remaining 2%. The radioactive
peak eluting at 5 min, asnoted above, isdependent on the preesnce Sf9
membranes. This peak is greater in the CYP2A4 than the CYP2A5 sample
illustrated in figure 4 since more CYP2A4 was used, and therefore more Sf9
membrane proteinsare present. The kinetic parametersfor the 3,4-epoxidation of
coumarin by CYP2A4 were determined. The Km was8.7 Æ 3.6 mm, the Vmax
0.20 Æ 0.04 nmol minÀ1 nmolÀ1 CYP. The product distribution did not change
with coumarin concentration.
The major product of CYP2A5-catalysed coumarin metabolism detected was
7-hydroxycoumarin (figure 4B), consistent with the previous characterizations of
2A5 as a coumarin 7-hydroxylase (Negishi et al. 1989, Felicia et al. 2000). Small
amountsof three other metabolites, o-HPA, 6-hydroxy and 8-hydroxy coumarin,
were also detected (figure 4B). Compared with the kinetic parameters of coumarin
7-hydroxylation by CYP2A5, CYP2A4-catalysed coumarin 3,4-epoxidation has a
ninefold higher Km and a 40-fold lower Vmax (Felicia et al. 2000). Relative to

Coumarin metabolism by CYP2A enzymes
79
Figure 4. Radioflow HPLC analysis of coumarin metabolites. 14C-coumarin (5 mm) wasincubated for
30 min with 33.9 pmol CYP2A4 (A) or 1.8 pmol CYP2A5 (B) asdescribed in the Materialsand
methods. The products of the reaction were co-injected with standards on a C18 HPLC column.
CYP2A5-catalysed coumarin 7-hydroxylation, CYP2A4 is not a very efficient
catalyst of coumarin 3,4-epoxidation. This is also true compared to the rates of
coumarin 3,4-epoxidation by the rat and human P450sCYP1A1, CYP1A2 and
CYP2E2 (Born et al. 2002). Recombinant human CYP1A1 and rat CYP1A2
catalyse coumarin 3,4-epoxidation at a rate of 6.26 and 22.2 nmol minÀ1 nmolÀ1
CYP, respectively (Born et al. 2002). These rates are 31- and 111-fold higher than
the rate of CYP2A4-mediated coumarin 3,4-epoxidation.
Coumarin 3,4-epoxide isreactive and could bind to cellular protein. We
determined here that 3,4-epoxidation isthe major pathway of coumarin metabo-
lism catalysed by CYP2A4. Negishi et al. (1989) reported that even though
CYP2A4 did not metabolize coumarin by 7-hydroxylation, coumarin did inhibit
CYP2A4-mediated testosterone 15ꢀ-hydroxylation. Both enzyme modification by
coumarin 3,4-epoxide and competitive inhibition may account for the inhibition
observed by Negishi et al. (1989).
The striking difference in the efficiency of coumarin 7-hydroxylation by
CYP2A4 and CYP2A5 has been extensively studied by Honkakoski and Negishi
(1997), Lindberg and Negishi (1989) and Negishi et al. (1989). By using site-
directed mutagenesis, they changed three amino acids in CYP2A4 to their

80
L. B. von Weymarn and S. E. Murphy
counterparts in CYP2A5. The resulting enzyme had coumarin 7-hydroxylase
activity of equal efficiency to that of CYP2A5 (Lindberg and Negishi 1989).
The mutated amino acidswere 117, 209 and 365. These three amino acidshave
been indicated to play a role in coumarin metabolism by several CYP2A enzymes
(Honkakoski and Negishi 1997). The CYP2A enzymes capable of catalysing the 7-
hydroxylation of coumarin all have a phenylalanine at position 209. CYP2A4,
which is not a coumarin 7-hydroxylase, has a leucine at position 209. CYP2A13
and CYP2A4 both have an alanine at position 117 compared with valine in 2A5
and 2A6, which may contribute to the low coumarin metabolism observed with
2A4 and 2A13. With respect to amino acids 117, 209 and 365, CYP2A13 is
identical to rat CYP2A3. CYP2A3 isan efficient coumarin 7-hydroxylaes (von
Weymarn et al. 1999). However, CYP2A3, like CYP2A13, also catalyses the 3,4-
epoxidation of coumarin.
A number of CYP2A enzymes in different species have sequence homology
>85% (Honkakoski and Negishi 1997), however their substrate specificities differ
significantly. This is clearly illustrated by the differences observed in coumarin
and NNK metabolism by CYP2A4, CYP2A5, CYP2A6 and CYP2A13. CYP2A6
and CYP2A13 are 94% homologous, they differ by only 32 amino acids (Su et al.
2000). We report here that compared with CYP2A6, CYP2A13 isnot a specific
coumarin 7-hydroxylase. Su et al. (2000) reported that CYP2A13 efficiently
metabolizes NNK, however 2A6 is significantly less efficient at catalysing NNK
metabolism (Patten et al. 1996). A different relationship exists between the closely
related CYP2A enzymes in the mouse. CYP2A4 is not a catalyst of coumarin 7-
hydroxylation and isa poor catalyst of NNK hydroxylation while CYP2A5 isa
very efficient catalyst of both reactions.
While amino acid residues 117, 309 and 365 are clearly important in determin-
ing the substrate specificity of CYPs 2A4 and 2A5, other amino acids must play a
role in CYP2A13 and other CYP2A enzymes. Complete characterization of
coumarin metabolism by CYP2A enzymes should help to identify amino acid
residues important in defining the active site of these enzymes. This information,
in turn, should contribute to our understanding of the role specific amino acids
play in determining the specificity of nitrosamine metabolism by these enzymes.
Acknowledgements
The authorsthank Xinxin Ding for providing CYP2A13. The tsudy was
supported by Grant CA-74913 from the National Cancer Institute.
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