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
Trang 1O-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 Kim1, Emre M Isin2, Chul-Ho Yun1, Dong-Hyun Kim3 and F P Guengerich2
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
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: f.guengerich@vanderbilt.edu
Note
The conventions used for kinetic hydrogen
isotope effects are D k ¼ intrinsic kinetic
deuterium isotope effect,DV ¼ H
k cat ⁄ D
k cat , and D (V ⁄ K) ¼ ( H kcat⁄ H Km) ⁄ ( D kcat⁄ D Km) [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
b5, cytochrome b5(b5, 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]).
Trang 2natural 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.
Trang 3mixture 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 d17-OEt coumarin and d27-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 d2 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)2¼ 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)
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 d0and 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 kcatand Km 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
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 d2⁄ d 0 formaldehyde product b A 1 : 1 molar mixture of d0and [1-ethyl-d2] 7-OEt coumarin was used as the substrate, and the kinetic isotope effect was measured from the ratio of d1⁄ d 0 acetaldehyde product c [Methyl-d2] 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-d1] 7-OEt coumarin was used as the substrate, and the kinetic isotope effect was measured from the ratio of d1⁄ d 0 acetaldehyde product.
Trang 4Fig 2 Steady-state kinetics of 7-OR coumarin O-dealkylation by P450 1A2 (A) 7-OEt coumarin Steady-state experiments were performed with d0(l) and 1,1-d2-ethyl (k) substrates (B) 7-OMe coumarin Steady-state experiments were performed with d0(l) and O-methyl d3(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
kcat
(min)1)
Km (l M ) kcat⁄ K m DV D (V ⁄ K)
kcat (min)1)
Km (l M ) kcat⁄ K m DV 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
d2 0.25 ± 0.02 12 ± 4 0.02 ± 0.01 15 ± 1 24 ± 5 0.64 ± 0.13
2E1 d0 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).bApparent at [S] ¼ 400 l M
Trang 5when 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 (kcat 0.45 min)1)
but too low to obtain reliable measurements for the
intramolecular and intermolecular competitive
values DV¼ 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
kcat(min)1) Km(l M ) kcat⁄ K m DV D (V ⁄ K) kcat(min)1) Km(l M ) kcat⁄ K m DV 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
d3 0.23 ± 0.02 13 ± 7 0.016 ± 0.006 7.3 ± 0.3 11 ± 2 0.65 ± 0.12
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 kcat(min)1) Km(l M ) kcat⁄ K m DV D (V ⁄ K) kcat(min)1) Km(l M ) kcat⁄ K m DV 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
d2 0.24 ± 0.01 6.7 ± 1.7 0.036 ± 0.009 14 ± 1 15 ± 2 0.90 ± 0.10
7-OMe d0 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
Trang 6liver microsomes at lower 7-OEt coumarin
concentra-tions [23] At very high concentraconcentra-tions of substrate, a
lower isotope effect might be expected due to the
con-tribution of P450 2E1, an enzyme with a higher Km
[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 Kmbut not kcat [32,33] This possibility
has not been evaluated with 7-OR coumarins and P450
due to the lower rates and the difficulty of estimating
kcatand Km (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-H2O 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
Vb(nmol product ⁄ nmol P450 min)1) D V
Vb(nmol product ⁄ nmol P450 min)1) D V
a Used at concentration of 100 l M in all experiments b Results are expressed as means of duplicate experiments, which differed < 10%.
Trang 7tions of the procedures described elsewhere [38] Insect cell
microsomes from P450 1A2⁄ baculovirus infections
(Super-somes) were purchased from BD Gentest (Woburn, MA,
USA)
Rat NADPH-P450 reductase was expressed in E coli
and purified as described [39]
Human b5 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 b5, 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, 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 CH3CN and diluted into enzyme
reactions, with final organic solvent concentrations < 1%
(v⁄ v)
Incubations were generally performed for 5–10 min at
37C, terminated with 0.10 mL of 17% HClO4, and
centri-fuged (103 g, 10 min) CH2Cl2 (1.0 mL) was added to the
supernatant to extract the products followed by
centrifuga-tion at 103g(process repeated one more time) The organic
layers were combined, and the CH2Cl2was removed under
a N2 stream The products, 7-hydroxy coumarin and
3-hydroxy, 7-OR coumarin, were analyzed by HPLC using
a Toso ODS-80TM octadecylsilane (C18) column (4.6 mm
150 mm, 5 lm) with the mobile phase H2O:CH3CN
(55 : 45, v⁄ v) containing 10 mm HClO4, a flow rate of
1.0 mLÆmin)1, and monitoring at A330[19] Kinetic
parame-ters (Km and kcat) 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 (C18) column (6.2 mm· 80 mm,
3 lm) with a mobile phase of H2O:CH3CN (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 mLÆmin)1 directed to the mass spectrometer Deuterium incorporation was deter-mined using negative ion atmospheric pressure chemical ionization MS (source temperature 550C, heated capillary temperature 180C, heated capillary voltage )20 V, tube lens voltage )40 V, ionization current 5 lA, sheath gas (N2) pressure 70 psi, auxiliary gas (N2) 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-d1]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 (A330) for reactions of purified, reconstituted P450 1A2 and 2E1 with 7-OEt coumarin (d0and d2)
Fig S2 HPLC traces (A330) for reactions using five human liver microsomal samples with 7-OEt coumarin (d0 and d2) A blank reaction (no NADPH) is shown for d2subsrate
This material is available as part of the online article from http://www.blackwell-synergy.com