Báo cáo khoa học: Kinetic deuterium isotope effects for 7-alkoxycoumarin O-dealkylation reactions catalyzed by human cytochromes P450 and in liver microsomes Rate-limiting C-H bond breaking in cytochrome P450 1A2 substrate oxidation pdf

Kinetic deuterium isotope effects for 7-alkoxycoumarin
O-dealkylation reactions catalyzed by human cytochromes
P450 and in liver microsomes
Rate-limiting C-H bond breaking in cytochrome P450 1A2 substrate
oxidation
Keon-Hee Kim
1
, Emre M. Isin
2
, Chul-Ho Yun
1
, Dong-Hyun Kim
3
and F. P. Guengerich
2
1 Hormone Research Center and School of Biological Sciences and Technology, Chonnam National University, Gwangju, Korea
2 Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, TN, USA
3 Doping Control Center, Korean Institute of Science and Technology, Seoul, Korea
Cytochrome P450 (P450, EC 1.14.14.1) enzymes
catalyze the oxidation of a great variety of steroids,
fat-soluble vitamins, eicosanoids, and numerous xeno-
biotic chemicals including drugs, natural products,
carcinogens, pesticides, and other compounds [4,5].
The study of these enzymes has been facilitated by the
availability of artificial substrates that can be utilized
as probes because of their spectral and fluorescent
properties. Among these are the coumarins [6,7] and
resorufins [7,8]. Coumarin and several derivatives are
Keywords
alkoxycoumarins; coumarins; Cytochrome
P450; kinetic isotope effects; microsomal

reactions
Correspondence
F. P. Guengerich, Department of
Biochemistry and Center in Molecular
Toxicology, Vanderbilt University School of
Medicine, Nashville, TN 37232–0146, USA
Fax: +615 322 3141
Tel.: +615 322 2261
E-mail:
Note
The conventions used for kinetic hydrogen
isotope effects are
D
k ¼ intrinsic kinetic
deuterium isotope effect,
D
V ¼
H
k
cat
⁄
D
k
cat
,
and
D
(V ⁄ K) ¼ (
H
k

cat
⁄
H
K
m
) ⁄ (
D
k
cat
⁄
D
K
m
) [2,3].
(Received 20 February 2006, accepted
17 March 2006)
doi:10.1111/j.1742-4658.2006.05235.x
7-Ethoxy (OEt) coumarin has been used as a model substrate in many cyto-
chrome P450 (P450) studies, including the use of kinetic isotope effects to
probe facets of P450 kinetics. P450s 1A2 and 2E1 are known to be the
major catalysts of 7-OEt coumarin O-deethylation in human liver micro-
somes. Human P450 1A2 also catalyzed 3-hydroxylation of 7-methoxy
(OMe) coumarin at appreciable rates but P450 2E1 did not. Intramolecular
kinetic isotope effects were used as estimates of the intrinsic kinetic deuter-
ium isotope effects for both 7-OMe and 7-OEt coumarin dealkylation reac-
tions. The apparent intrinsic isotope effect for P450 1A2 (9.4 for
O-demethylation, 6.1 for O-deethylation) showed little attenuation in other
competitive and noncompetitive experiments. With P450 2E1, the intrinsic
isotope effect (9.6 for O-demethylation, 6.1 for O-deethylation) was attenu-
ated in the noncompetitive intermolecular experiments. High noncompeti-

tive intermolecular kinetic isotope effects were seen for 7-OEt coumarin
O-deethylation in a baculovirus-based microsomal system and five samples
of human liver microsomes (7.3–8.1 for O-deethylation), consistent with the
view that P450 1A2 is the most efficient P450 catalyzing this reaction in
human liver microsomes and indicating that the C-H bond-breaking step
makes a major contribution to the rate of this P450 (1A2) reaction. Thus,
the rate-limiting step appears to be the chemistry of the breaking of this
bond by the activated iron-oxygen complex, as opposed to steps involved in
the generation of the reactive complex. The conclusion about the rate-limit-
ing step applies to all of the systems studied with this model P450 1A2 reac-
tion including human liver microsomes, the most physiologically relevant.
Abbreviations
b
5
, cytochrome b
5
(b
5
, 1.6.2.2); di-12 : 0 GPC, L-a-dilauroyl-sn-glycero-3-phosphocholine; MS, mass spectrometry; OEt, ethoxy; OMe,
methoxy; OR, alkoxy; P450, cytochrome P450 (also termed ‘heme-thiolate protein P450’ [1]).
FEBS Journal 273 (2006) 2223–2231 ª 2006 The Authors Journal compilation ª 2006 FEBS 2223
natural products themselves, but the artificial 7-alkoxy
derivatives have been particularly useful because of the
large fluorescent changes that occur upon hydroxyla-
tion of the alkyl group and release to form 7-hydroxy-
coumarin (umbelliferone) [6,7].
Kinetic hydrogen isotope effects, especially with deu-
terium, have been used to probe aspects of the cata-
lytic mechanisms of many enzymes, including P450s
[9–12]. A basic concept is that the existence of a non-

competitive intermolecular kinetic deuterium isotope
effect argues that the C-H bond-breaking step is at
least partially rate-limiting [2,3,13]. If an alternate
reaction path is possible, then the impedance of one
pathway by deuterium substitution may yield a ‘meta-
bolic switch’ (or ‘isopically sensitive branching’) [14] to
produce enhanced levels of the other product, although
this is not always the case due to kinetic issues. Both
high and low kinetic deuterium isotope effects have
been reported for various P450 reactions. Although
early work in the field suggested that high isotope
effects were uncommon with P450s, particularly in
microsomes [9,15,16], in more recent work a case may
be made that a significant contribution of the C-H
bond-breaking step to overall rates of catalysis may be
more the norm than the exception, at least in purified
enzyme systems [17]. For instance, kinetic deuterium
isotope effects as high as 15 have been reported with
rabbit P450 1A2 [18].
Human P450 2A6 showed high intramolecular kin-
etic isotope effects for the O-dealkylation of 7-OMe
and 7-OEt coumarin (10 and 6, respectively) (Fig. 1),
which were not attenuated in noncompetitive experi-
ments, arguing that C-H bond breaking is a rate-limit-
ing step [19]. However, P450 2A6 does not make a
substantial contribution to this particular enzyme
activity in liver microsomes, and the question can be
raised as to whether the microsomal (and in vivo) rates
are limited by reduction and other steps involved in
the generation of the reactive oxygen species, as

opposed to the reactions of the activated complex with
substrates, in light of the low endogenous concentra-
tion of NADPH-P450 reductase [20]. In earlier work,
the laboratory of Lu and Miwa [10,21,22] reported
high intrinsic kinetic deuterium isotope effects for
7-OEt coumarin O-deethylation by some rat P450
enzymes (today known as P450s 2B1 and 1A1). How-
ever, these high isotope effects were strongly attenu-
ated in either intermolecular noncompetitive studies
with the purified enzymes or with liver microsomes,
except in the case of liver microsomes prepared from
3-methylcholanthrene-treated hamsters (in this case the
enzyme was not purified) [22].
We analyzed two human liver P450s implicated in
7-OEt coumarin O-deethylation, namely P450s 1A2
and 2E1 [23]. 7-OMe coumarin was also used as a
deuterated probe, to avoid the issue of prochirality
inherent in the ethyl group. We found high isotope
effects expressed even in the noncompetitive experi-
ments performed in microsomes. A major fraction of
an alternate product, derived from 3-hydroxylation,
was found with these enzymes and in microsomes. The
results are interpreted in the context of a rate-limiting
chemical step involved in the reaction of the activated
enzyme complex (Fe-O species) with the substrate,
even in the most relevant biological system, human
liver microsomes.
Results and discussion
Intramolecular kinetic isotope effects
The intramolecular kinetic isotope effects measure the

comparative rates for cleavage of a C-H bond and a
C-D bond at a single carbon [3]. The value obtained
from such an experiment can be used as an approxi-
mation of the true intrinsic kinetic deuterium isotope
effect and for comparison with all other types of
experiments to ascertain the extent to which any
attenuation has occurred [3]. The intermolecular
experiments, the studies which provide the most infor-
mation about the rate-limiting steps (see below), must
be considered in the context of the estimated intrinisic
isotope effects.
The values for 7-OMe O-demethylation, measured
by MS, were 9.4 and 9.6 for P450s 1A2 and 2E1,
respectively (Table 1). The corresponding values were
6.1 for both P450s 1A2 and 2E1 with 7-OEt coumarin
(Table 1). Whether these values should necessarily be
identical to each other (for the two enzymes) is not
clear. In one sense they should be independent of what
the rate-limiting steps are for these two enzymes (and
P450 2A6 [19]), if similar mechanisms are operative.
At least two factors can perturb these values so that
they are not true intrinsic kinetic deuterium isotope
effects. One is the issue of prochirality in the case of
7-OEt coumarin. That is, the two methylene hydrogens
are not equivalent, and we are using a racemic
Fig. 1. Oxidation reactions with coumarins catalyzed by P450s.
P450 coumarin dealkylation isotope effects K H. Kim et al.
2224 FEBS Journal 273 (2006) 2223–2231 ª 2006 The Authors Journal compilation ª 2006 FEBS
mixture. Conceivably the P450s being examined here
could show a partial or strict stereochemical preference

for one or the other face. For instance, partial selectiv-
ity has been seen in oxidations of ethylbenzene [24]
and a substituted nitrosamine [25]. If strict stereoselec-
tivity of R versus S hydrogens occurs with 7-OEt
coumarin, then the experiment effectively becomes an
intermolecular competitive experiment (see below).
Studies with 7-OMe coumarin have an advantage in
that there is no issue with pro-chirality. However, with
both d
1
7-OEt coumarin and d
2
7-OMe coumarin some
perturbation can occur because of a geminal secondary
kinetic isotope effect. These values are traditionally
£ 1.2 for each deuterium atom [26], and only limited
literature is available about P450 (gem) secondary
kinetic isotope effects [19,27–29]. The existence of a
gem secondary kinetic isotope effect has the effect of
attenuating the rate of C-H bond-breaking, so in prin-
ciple the intrinsic kinetic isotope effect would be even
higher than that estimated by this method (assuming a
secondary isotope effect > 1). With d
2
7-OMe couma-
rin, the secondary effects would be multiplicative [26],
and if a secondary isotope effect as high as 1.2 existed,
it would rise to (1.2)
2
¼ 1.44. Although we cannot

readily estimate the secondary isotope effect, the values
of the intramolecular values in Table 1 are reasonable
but should be considered lower estimates of the intrin-
sic kinetic deuterium isotope effects.
Intermolecular competitive kinetic isotope effects
The intermolecular competitive kinetic isotope effects
were estimated using MS of the products (Table 1).
With 7-OEt coumarin, the value was high for
P450 1A2 (9.5); with 7-OMe coumarin the value for
P450 1A2 was 10.6 and for P450 2E1 was 7.7. The
value of these measurements lies in their comparison
with the values measured in the intramolecular experi-
ments, i.e. the estimates of the intrinsic kinetic isotope
effects (Table 1, see above). The value for P450 1A2
was not diminished, within experimental error and
considering the caveats about secondary isotope effects
mentioned above, but some attenuation was seen for
P450 2E1, from 7.7 (± 0.5) to 4.2 (± 1.0) (Table 1).
Attenuation of an intrinsic isotope effect (estimated
with the intramolecular experiment in Table 1) in a
competitive experiment provides evidence that the rate
of exchange of substrates is a slow process relative to
forward progress past an irreversible step. That is, if
an isotope effect is sensed in the enzyme, the enzyme
will process the deuterated substrate if exchanging that
(deuterated) substrate for a protiated one takes longer
(than the C-H bond cleavage process).
Non-competitive intermolecular isotope effects
The noncompetitive intermolecular isotope effects were
measured by running assays with d

0
and perdeuterated
(at the carbon being oxidized) substrates and compar-
ing the v versus S plots (Figs 2 and 3, and Tables 2
and 3).
The patterns were very similar for 7-OMe and
7-OEt coumarin, with P450 1A2 showing high isotope
effects (8–16) and P450 2E1 yielding isotope effects
of 2–4, allowing for the variability in both cases. For
P450 2E1, the discernment of k
cat
and K
m
components
was difficult (Fig. 3).
With P450 1A2, 3-hydroxylation was observed
(Tables 2 and 3), with the catalytic efficiency being
comparable to that of O-dealkylation (see Supplement-
ary material). These products were also observed in
reactions with P450 2A6 [19] although with less effi-
ciency than O-dealkylation. (3-Hydroxylation has also
been reported in human [30] and other [31] liver micro-
somes, but rates were not reported.)
As shown in Figs 2 and 3 and Tables 2 and 3, deu-
teration of the alkoxy group had little tendency to
divert the reaction to 3-hydroxylation (Fig. 1), even
Table 1. Kinetic isotope effects for 7-OR coumarin O-dealkylation by purified P450s estimated by MS.
Kinetic isotope effect
Intermolecular competitive Intramolecular (noncompetitive)
P450 7-OMe coumarin

a
7-OEt coumarin
b
7-OMe coumarin
c
7-OEt coumarin
d
1A2 10. 6 ± 0.4 9.5 ± 2.0 9.4 ± 0.4 6.1 ± 1.4
2E1 7.7 ± 0.5 4.2 ± 1.0 9.6 ± 0.2 6.1 ± 1.1
a
A 1 : 1 molar mixture of d
0
and [methyl-d
3
] 7-OMe coumarin was used as the substrate, and the kinetic isotope effect was measured from
the ratio of d
2
⁄ d
0
formaldehyde product.
b
A 1 : 1 molar mixture of d
0
and [1-ethyl-d
2
] 7-OEt coumarin was used as the substrate, and the
kinetic isotope effect was measured from the ratio of d
1
⁄ d
0

acetaldehyde product.
c
[Methyl-d
2
] 7-OMe coumarin was used as the substrate,
and the kinetic isotope effect was measured from the ratio of d
2
⁄ d
1
formaldehyde product (multiplied by 2 for the statistical effect).
d
[1-Ethyl-d
1
] 7-OEt coumarin was used as the substrate, and the kinetic isotope effect was measured from the ratio of d
1
⁄ d
0
acetaldehyde
product.
K H. Kim et al. P450 coumarin dealkylation isotope effects
FEBS Journal 273 (2006) 2223–2231 ª 2006 The Authors Journal compilation ª 2006 FEBS 2225
Fig. 2. Steady-state kinetics of 7-OR coumarin O-dealkylation by P450 1A2. (A) 7-OEt coumarin. Steady-state experiments were performed
with d
0
(l) and 1,1-d
2
-ethyl (k) substrates. (B) 7-OMe coumarin. Steady-state experiments were performed with d
0
(l)andO-methyl d
3

(k)
substrates. The formation of 7-OH coumarin was measured using HPLC. See Table 2 for parameters.
Fig. 3. Steady-state kinetics of 7-OR coumarin O-dealkylation by P450 2E1. (A) 7-OEt coumarin. Steady-state experiments were performed
with d
0
(s) and 1,1-d
2
-ethyl (d) substrates. (B) 7-OMe coumarin. Steady-state experiments were performed with d
0
(l) and O-methyl d
3
(k)
substrates. The formation of 7-OH coumarin was measured using HPLC. See Table 2 for parameters.
Table 2. Rates of 7-OEt coumarin O-deethylation and 3-hydroxylation and intermolecular noncompetitive isotope effects.
P450
7-OEt
coumarin
substrate
O-Deethylation 3-Hydroxylation
k
cat
(min
)1
)
K
m
(lM) k
cat
⁄ K
m

D
V
D
(V ⁄ K)
k
cat
(min
)1
)
K
m
(lM) k
cat
⁄ K
m
D
V
D
(V ⁄ K)
1A2 d
0
2.0 ± 0.1 6 ± 1 0.34 ± 0.05 8.0 ± 1.0 16 ± 6 14 ± 1 27 ± 6 0.51 ± 0.11 0.92 ± 0.08 0.81 ± 0.25
d
2
0.25 ± 0.02 12 ± 4 0.02 ± 0.01 15 ± 1 24 ± 5 0.64 ± 0.13
2E1 d
0
1.6 ± 0.2 480 ± 90 0.0033 ± 0.0007 2.2 ± 0.9  4
a
0.95

b
d
2
0.7 ± 0.3 1300 ± 700 0.00057 ± 0.00004
a
Estimated from slopes of v versus S plots (Fig. 3A).
b
Apparent at [S] ¼ 400 lM.
P450 coumarin dealkylation isotope effects K H. Kim et al.
2226 FEBS Journal 273 (2006) 2223–2231 ª 2006 The Authors Journal compilation ª 2006 FEBS
when high kinetic isotope effects occurred. This result
is in contrast to the case of 7-OMe coumarin with
P450 2A6 [19] (but not 7-OEt coumarin [19]). Harada
et al. [21] reported trace 6-hydroxylation of 7-OEt cou-
marin in rat liver microsomes and a strong switch to
this alternate product as a result of deuterium
substitution. No other products were detected in this
work (see Supplementary material).
In other experiments which are not presented, recom-
binant human P450 1A1 was used in some experiments,
because some work had shown catalytic activity toward
7-OEt coumarin [23]. Rates for 7-OMe coumarin
O-demethylation were very low ( 0.02 min
)1
); 7-OEt
O-deethylation rates were higher (k
cat
0.45 min
)1
)

but too low to obtain reliable measurements for the
intramolecular and intermolecular competitive experi-
ments. For 7-OEt coumarin O-deethylation, the
values
D
V ¼ 3.0 ± 0.2 and
D
(V ⁄ K) ¼ 2.5 ± 0.9 were
obtained. The rate of formation of 3-hydroxy, 7-OEt
coumarin was 10-fold slower.
Apparent intermolecular noncompetitive kinetic
isotope effects in human liver microsomes and
a baculovirus-based recominant system with
over-expressed NADPH-P450 reductase
One possibility to consider is that the rate-limiting nat-
ures of reaction steps are perturbed in systems invol-
ving purified P450 enzymes that are reconstituted with
more NADPH-P450 reductase (EC 1.6.2.4) than is
normally associated with the P450s in microsomal
membranes [20]. Relatively few studies have considered
comparisons because of the issue of multiple P450s cat-
alyzing a reaction of interest in microsomes. Miwa
et al. [22] reported lack of attenuation of the kinetic
isotope effect for 7-OEt coumarin O-deethylation in
noncompetitive studies with liver microsomes prepared
from 3-methylcholanthrene-treated hamsters, which at
that time was considered unusual in that the isotope
effects were not well-expressed in other systems. In pre-
vious work in this laboratory we analyzed rat P450 1A2
and found similar values for the isotope effects for

7-OMe resorufin O-demethylation, considered to be a
relatively selective P450 1A2 substrate, with the purified
enzyme and microsomes (assuming that other P450s do
not catalyze this reaction at appreciable rates) [18].
The high noncompetitive isotope effects seen in the
systems comprised of purified enzymes were also seen
in insect cell microsomes from a baculovirus-based
expression system in which NADPH-P450 reductase is
over-expressed (Table 4). The high values for noncom-
petitive intermolecular kinetic isotope effects for the
O-dealkylation of both 7-OMe and 7-OEt coumarin
are similar to those measured with the reconstituted
systems (Tables 2 and 3).
High noncompetitive isotope effects were also seen
in human liver microsomes (Table 5, Supplementary
material). These results are consistent with the earlier
conclusions that P450 1A2 is the major enzyme
involved in 7-OEt coumarin O-deethylation in human
Table 3. Rates of 7-OMe coumarin O-demethylation and 3-hydroxylation and intermolecular noncompetitive isotope effects.
P450
7-OMe
coumarin
substrate
O-Demethylation 3-Hydroxylation
k
cat
(min
)1
) K
m

(lM) k
cat
⁄ K
m
D
V
D
(V ⁄ K) k
cat
(min
)1
) K
m
(lM) k
cat
⁄ K
m
D
V
D
(V ⁄ K)
1A2 d
0
4.1 ± 0.3 31 ± 8 0.13 ± 0.04 15 ± 2 8.0 ± 4.0 7.7 ± 0.4 22 ± 4 0.35 ± 0.06 1.1 ± 0.1 0.54 ± 0.14
d
3
0.23 ± 0.02 13 ± 7 0.016 ± 0.006 7.3 ± 0.3 11 ± 2 0.65 ± 0.12
2E1 d
0
 2.8  3

a
 1.1
d
3
a
See Fig. 3(B).
Table 4. Rates of 7-OR coumarin O-dealkylation and 3-hydroxylation and intermolecular noncompetitive isotope effects using human P450
1A2 and NADPH-P450 reductase expressed in a baculovirus-based system.
Alkoxy
coumarin
O-Dealkylation 3-Hydroxylation
Substrate k
cat
(min
)1
) K
m
(lM) k
cat
⁄ K
m
D
V
D
(V ⁄ K) k
cat
(min
)1
) K
m

(lM) k
cat
⁄ K
m
D
V
D
(V ⁄ K)
7-OEt d
0
2.0 ± 0.1 8.0 ± 2.0 0.25 ± 0.06 8.3 ± 0.5 6.9 ± 2.4 11 ± 1 11 ± 1 1.0 ± 0.1 0.79 ± 0.09 1.1 ± 0.2
d
2
0.24 ± 0.01 6.7 ± 1.7 0.036 ± 0.009 14 ± 1 15 ± 2 0.90 ± 0.10
7-OMe d
0
4.1 ± 0.1 5.0 ± 0.7 0.82 ± 0.12 10 ± 1 6 ± 2 13 ± 1 33 ± 2 0.40 ± 0.04 1.4 ± 0.20 0.60 ± 0.10
d
3
0.40 ± 0.02 3.0 ± 1.0 0.13 ± 0.04 9.0 ± 1.0 13 ± 2 0.70 ± 0.10
K H. Kim et al. P450 coumarin dealkylation isotope effects
FEBS Journal 273 (2006) 2223–2231 ª 2006 The Authors Journal compilation ª 2006 FEBS 2227
liver microsomes at lower 7-OEt coumarin concentra-
tions [23]. At very high concentrations of substrate, a
lower isotope effect might be expected due to the con-
tribution of P450 2E1, an enzyme with a higher K
m
[23] (Tables 2 and 3). The high noncompetitive isotope
effects are interpreted to mean that the step(s) invol-
ving the chemistry of C-H bond cleavage are rate-

limiting in the reaction even in liver microsomes, as
opposed to steps involving generation of the reactive
oxygenated iron species.
Conclusions
The human P450s that have been most implicated in
7-OEt coumarin oxidation were analyzed for kinetic
isotope effects in O-dealkylation, as well as with the
alternative substrate 7-OMe coumarin. P450 1A2
clearly showed the highest apparent intrinsic kinetic
isotope effect, which was not considerably attenuated
in various experimental systems, even in liver micro-
somes. These results indicate that the rate of C-H
bond-breaking is a major factor in determining the
rates of the reactions under all conditions. Some shift-
ing of the oxidations to the alternate 3-hydroxylation
reactions occurs with some of the enzymes (with
7-OMe and 7-OEt coumarin), but not to the extent to
utilize all of the electrons that are delivered into the
P450 system.
P450 2E1 did not show full expression of the isotope
effect, and the possibility of rate-limiting steps other
than C-H bond breaking is suggested. Earlier work
with human P450 2E1-catalyzed oxidations of ethanol
and acetaldehyde demonstrated rate-limiting steps fol-
lowing product formation and a resulting kinetic iso-
tope effect on K
m
but not k
cat
[32,33]. This possibility

has not been evaluated with 7-OR coumarins and P450
due to the lower rates and the difficulty of estimating
k
cat
and K
m
(Fig. 3). Another possible complication is
the oxidation of the product acetaldehyde by P450 2E1
[33] and the effect on the apparent kinetic constants,
which has not been considered in detail here because
the primary phenolic products are being measured.
To summarize, a major conclusion of the work per-
formed with several types of kinetic isotope effects is
that P450 1A2 is a major catalyst of the model alkoxy-
coumarin reactions in human liver. The actual sub-
strate oxidation step is rate-limiting, as opposed to
steps involved in the generation of the reactive
enzyme-oxygen complex, even in the microsomal sys-
tem, and these results should apply to in vivo consider-
ations. The conclusions may apply to other P450
reactions, at least to those catalyzed by P450 1A2.
Experimental procedures
Chemicals
7-OMe and 7-OEt coumarin were purchased from Sigma-
Aldrich (Milwaukee, WI, USA) and recrystallized from
EtOH-H
2
O mixtures before use. The deuterated substrates
were prepared and characterized as described elsewhere
[19]. The syntheses and characterization of 3-hydroxy,

7-OMe coumarin and 3-hydroxy, 7-OEt coumarin are
described elsewhere [19].
Enzymes
Human liver samples were obtained through Tennessee
Donor Services, stored at )80 °C, and used to prepare
microsomal samples [34].
Human P450s 1A2 [35], 2E1 [36], and 1A1 [37] were
expressed in Escherichia coli and purified using modifica-
Table 5. Rates of 7-OEt coumarin O-deethylation and intermolecular noncompetitive isotope effects in human liver microsomes.
Sample
code no.
7-OEt
coumarin
substrate
a
O-Deethylation 3-Hydroxylation
V
b
(nmol product ⁄
nmol P450 min
)1
)
D
V
V
b
(nmol product ⁄
nmol P450 min
)1
)

D
V
104 d
0
0.048 8.1 0.018 0.95
d
2
0.0059 0.019
109 d
0
0.25 8.1 0.17 1.0
d
2
0.31 0.17
110 d
0
0.14 7.8 0.077 0.70
d
2
0.18 0.11
115 d
0
0.17 7.1 0.12 0.86
d
2
0.024 0.14
132 d
0
0.19 7.3 0.093 0.66
d

2
0.026 0.14
a
Used at concentration of 100 lM in all experiments.
b
Results are expressed as means of duplicate experiments, which differed < 10%.
P450 coumarin dealkylation isotope effects K H. Kim et al.
2228 FEBS Journal 273 (2006) 2223–2231 ª 2006 The Authors Journal compilation ª 2006 FEBS
tions of the procedures described elsewhere [38]. Insect cell
microsomes from P450 1A2 ⁄ baculovirus infections (Super-
somes
Ò
) were purchased from BD Gentest (Woburn, MA,
USA).
Rat NADPH-P450 reductase was expressed in E. coli
and purified as described [39].
Human b
5
was expressed in E. coli JM109 cells from a
plasmid (pSE420 (Amp)) provided by S Asaki (Takeda
Pharmaceutical, Osaka, Japan). The protein was purified to
electrophoretic homogeneity using modifications of the
DEAE-cellulose and other chromatography methods des-
cribed elsewhere [19,40].
Enzyme assays
Typical steady-state coumarin oxidation reactions included
50 pmol P450, 100 pmol of NADPH-P450 reductase,
50 pmol of b
5
, and 30 lg of di-12 : 0 GPC in 0.50 mL

of 50 mm potassium phosphate buffer (pH 7.4) along with
a specified amount of the coumarin substrate. In some
cases, human liver microsomes (50 pmol P450 in 0.50 mL
of 50 mm potassium phosphate buffer, pH 7.4) or insect
cell microsomes from a baculovirus-based system contain-
ing human P450 1A2 and an excess of NADPH-P450
reductase (BD Gentest Supersomes
Ò
, 20 pmol P450 in
0.50 mL of 50 mm potassium phosphate buffer, pH 7.4)
were used instead of the recombinant (bacterial) P450 sys-
tem. An aliquot of an NADPH-generating system was used
to start reactions (final concentrations, 10 mm glucose 6-
phosphate, 0.5 mm NADP
+
, and 1 IU yeast glucose 6-
phosphate per mL [34]). 7-OMe and 7-OEt coumarin stocks
(50 mm) were made in CH
3
CN and diluted into enzyme
reactions, with final organic solvent concentrations < 1%
(v ⁄ v).
Incubations were generally performed for 5–10 min at
37 °C, terminated with 0.10 mL of 17% HClO
4
, and centri-
fuged (10
3
g, 10 min). CH
2

Cl
2
(1.0 mL) was added to the
supernatant to extract the products followed by centrifuga-
tion at 10
3
g (process repeated one more time). The organic
layers were combined, and the CH
2
Cl
2
was removed under
aN
2
stream. The products, 7-hydroxy coumarin and
3-hydroxy, 7-OR coumarin, were analyzed by HPLC using
a Toso ODS-80
TM
octadecylsilane (C
18
) column (4.6 mm
150 mm, 5 lm) with the mobile phase H
2
O:CH
3
CN
(55 : 45, v ⁄ v) containing 10 m m HClO
4
, a flow rate of
1.0 mLÆmin

)1
, and monitoring at A
330
[19]. Kinetic parame-
ters (K
m
and k
cat
) were determined using nonlinear regres-
sion analysis with Graph-Pad prism software (Graph-Pad,
San Diego, CA, USA). See Supplementary material for
typical chromatograms.
Intermolecular competitive and intramolecular noncom-
petitive kinetic isotope effects were estimated by analysis of
the mass spectra of 2,4-dinitrophenylhydrazone derivatives,
using the calculation methods previously described [19,41].
The substrate concentration used in these experiments was
50 lm with P450 1A2 and 300 lm with P450 2E1. Mass
spectra were recorded using HPLC-MS methods in the
Vanderbilt facility with a Thermo-Finnigan TSQ 7000
instrument (Thermo-Finnigan, Sunnyvale, CA, USA) using
a Zorbax octadecylsilane (C
18
) column (6.2 mm · 80 mm,
3 lm) with a mobile phase of H
2
O:CH
3
CN (46 : 54, v ⁄ v)
and a flow rate of 2 mLÆmin

)1
. The flow was split after the
column to result in a flow rate of 1 mLÆ min
)1
directed to
the mass spectrometer. Deuterium incorporation was deter-
mined using negative ion atmospheric pressure chemical
ionization MS (source temperature 550 °C, heated capillary
temperature 180 °C, heated capillary voltage )20 V, tube
lens voltage )40 V, ionization current 5 lA, sheath gas
(N
2
) pressure 70 psi, auxiliary gas (N
2
) pressure 10 p.s.i).
Acknowledgements
This work was supported in part by the Korea Research
Foundation Grant (KRF-2004–005-E00015) and United
States Public Health Service grants R01 CA090426 and
P30 ES000267. We thank M. V. Martin and W. A.
McCormick for preparing some of the enzymes, M. W.
Calcutt for preparing 7-[2-d
1
]OEt coumarin, and K.
Trisler for assistance in preparation of the manuscript.
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Supplementary material
The following supplementary material is available
online:
Fig. S1. HPLC traces (A
330
) for reactions of purified,
reconstituted P450 1A2 and 2E1 with 7-OEt coumarin
(d
0
and d
2
).
Fig. S2. HPLC traces (A
330
) for reactions using five
human liver microsomal samples with 7-OEt coumarin
(d
0
and d
2
). A blank reaction (no NADPH) is shown
for d
2
subsrate.
This material is available as part of the online article
from
K H. Kim et al. P450 coumarin dealkylation isotope effects

FEBS Journal 273 (2006) 2223–2231 ª 2006 The Authors Journal compilation ª 2006 FEBS 2231