Mechanism-based inactivation of cytochromes P450 2B1 and P450 2B6 by n- propylxanthate

Article abstract of DOI:10.1021/tx9802421

n-Propylxanthate (nPX) inactivated the 7-ethoxy-4- (trifluoromethyl)coumarin (7-EFC) O-deethylation activity of purified, reconstituted rat hepatic P450 2B1 or human P450 2B6 in a mechanism-based manner. The inactivation followed pseudo-first-order kinetics and was entirely dependent on both NADPH and nPX. The maximal rate constant for inactivation of P450 2B1 at 30 °C was 0.2 min^-1. The apparent K(I) was 44 μM, and the half-time for inactivation was 4.1 min. Purified, reconstituted human P450 2B6 was also inactivated by nPX with a K(I) of 12 μM. The κ(inactivation) for P450 2B6 was 0.06 min^-1, and the t(1/2) was 11 min. Incubations of P450 2B1 with nPX and NADPH for 20 min resulted in a 75% loss in enzymatic activity and a concurrent 25% loss of the enzyme's ability to form a reduced CO complex. Little loss in the absolute spectrum of nPX- inactivated P450 2B1 was observed. With P450 2B6, an 83% loss in enzymatic activity and a 12% loss in the CO-reduced spectra were observed. The extrapolated partition ratio for nPX with P450 2B1 was 32. P450 2B1 could be protected from inactivation by nPX by adding an alternate substrate to the reaction mixture. Removal of unbound nPX by dialysis did not reverse the inactivation. The alternate oxidant iodosobenzene was able to partially restore enzymatic activity to nPX-inactivated P450 2B1 samples. A stoichiometry for labeling of 1.2:1 for binding of radiolabeled nPX metabolite to P450 2B1 was seen. These results indicated that nPX inactivated P450 2B1 and P450 2B6 in a mechanism-based manner. P450 2B1 was inactivated primarily by a nPX reactive intermediate that bound to the apoprotein.

Full text of DOI:10.1021/tx9802421

Chem. Res. Toxicol. 1999, 12, 317-322
317
Mech a n ism -Ba sed In a ctiva tion of Cytoch r om es P 450 2B1
a n d P 450 2B6 by n -P r op ylxa n th a te
Ute M. Kent,† Stanislav Yanev,‡ and Paul F. Hollenberg*,†
Department of Pharmacology, The University of Michigan, Ann Arbor, Michigan 48109, and
Department of Drug Toxicology, Institute of Physiology, Bulgarian Academy of Sciences, Bulgaria
Received November 4, 1998
n-Propylxanthate (nPX) inactivated the 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC) O-
deethylation activity of purified, reconstituted rat hepatic P450 2B1 or human P450 2B6 in a
mechanism-based manner. The inactivation followed pseudo-first-order kinetics and was
entirely dependent on both NADPH and nPX. The maximal rate constant for inactivation of
P450 2B1 at 30 °C was 0.2 min-1. The apparent KI was 44 µM, and the half-time for inactivation
was 4.1 min. Purified, reconstituted human P450 2B6 was also inactivated by nPX with a KI
of 12 µM. The kinactivation for P450 2B6 was 0.06 min-1, and the t1/2 was 11 min. Incubations of
P450 2B1 with nPX and NADPH for 20 min resulted in a 75% loss in enzymatic activity and
a concurrent 25% loss of the enzyme’s ability to form a reduced CO complex. Little loss in the
absolute spectrum of nPX-inactivated P450 2B1 was observed. With P450 2B6, an 83% loss in
enzymatic activity and a 12% loss in the CO-reduced spectra were observed. The extrapolated
partition ratio for nPX with P450 2B1 was 32. P450 2B1 could be protected from inactivation
by nPX by adding an alternate substrate to the reaction mixture. Removal of unbound nPX by
dialysis did not reverse the inactivation. The alternate oxidant iodosobenzene was able to
partially restore enzymatic activity to nPX-inactivated P450 2B1 samples. A stoichiometry for
labeling of 1.2:1 for binding of radiolabeled nPX metabolite to P450 2B1 was seen. These results
indicated that nPX inactivated P450 2B1 and P450 2B6 in a mechanism-based manner. P450
2B1 was inactivated primarily by a nPX reactive intermediate that bound to the apoprotein.
2-ethynylnaphthalene (10), 9-ethynylphenanthrene (11),
or secobarbital (12). Relatively little is known about the
physiological role of the human 2B homologue, P450 2B6,
although some studies suggest that P450 2B6 was
expressed at elevated levels in breast cancer (13). Studies
by Osborne et al. also suggested that an increase in the
level of C16 R-hydroxylation of 17â-estradiol metabolism
in breast tissue may be a biomarker of breast cancer risk
(14). P450 2B6 comprises only about 0.2% of the total
P450s in human liver microsomes and may not be
expressed in all human livers (15, 16). However, a recent
study indicated that P450 2B6 expression could be
induced by phenobarbital in all the human livers that
were screened with a polyclonal anti-2B6 antibody (17).
The wide variability in the reported detection levels may
be due to the low abundance of this isoform combined
with a lack of available detection reagents such as specific
antibodies, substrates, or inactivators. Recently, a mono-
clonal antibody to cDNA-expressed P450 2B6 has been
developed (18). With the use of recombinant vaccinia or
baculovirus P450 2B6 expression systems, it has been
shown that P450 2B6 can metabolize a number of
different substrates such as nicotine (19), aminochrysene
and 3-methoxy-4-aminoazobenzene (20), tamoxifen (21),
7-benzyloxyresorufin (22), 7-ethoxycoumarin (23), testos-
terone (24), RP73401 (25), and phenanthrene and diaz-
epam (18). Roberts et al. also demonstrated that P450
2B6 expressed in AHH-11 lymphoblastoid cells was
inactivated by 9-ethynylphenanthrene (26).
In tr od u ction
Liver microsomal cytochromes P450 are involved in the
metabolism of many drugs and carcinogens. P450 en-
zymes catalyze the metabolism of a wide variety of
structurally distinct substrates (1), and particular P450
isoforms are also able to metabolize a number of different
substrates (1-3). The catalytic mechanism appears to be
common to all P450s and involves a two-electron reduc-
tion of molecular oxygen to form a reactive oxygen
intermediate and water (1).
The three-dimensional structure of mammalian cyto-
chromes has not been determined. Current information
about the critical active site amino acid residues involved
in substrate binding and catalysis has come primarily
from site-directed mutagenesis studies or from observa-
tions with naturally occurring mutants (4, 5). Additional
insight into the active site structure has been gained from
examining the crystal structures of a number of bacterial
P450s (6-9). Mechanism-based inactivators that undergo
catalytic conversion to reactive intermediates that co-
valently bind to amino acid side chains have been used
to identify peptides within the active site and in some
instances have also been used to locate key amino acid
residues that are involved in substrate metabolism.
Particularly successful were studies with the 2B rat and
rabbit enzymes using acetylenic compounds such as
* To whom correspondence should be addressed: Department of
Pharmacology, Medical Science Research Bldg. III, 1150 W. Medical
Center Dr., Ann Arbor, MI 48109-0632. E-mail: phollen@umich.edu.
† The University of Michigan.
Alkyl xanthates (R-OCS2) have been identified as
potential mechanism-based inactivators of P450 2B1 in
‡ Bulgarian Academy of Sciences.
10.1021/tx9802421 CCC: $18.00 © 1999 American Chemical Society
Published on Web 03/12/1999

318 Chem. Res. Toxicol., Vol. 12, No. 4, 1999
Kent et al.
washed to remove Triton N101 with Tris-acetate (pH 8.0), 0.25%
cholate, 0.7 M NaCl, â-mercaptoethanol, 15 mM imidazole, and
40% glycerol. P450 2B6 was eluted with 100 mM potassium
phosphate buffer (pH 7.4) containing 0.7 M NaCl, 0.5 M
imidazole, and 40% glycerol. The fractions that contained P450
2B6 were pooled and dialyzed against two 1 L changes of 100
mM potassium phosphate buffer (pH 7.4) containing 1 mM
EDTA and 20% glycerol and stored at -80 °C.
rat liver microsomes. The microsomal metabolism of
ethylxanthate required O2 and NADPH and could be
blocked by CO, metyraprone, and SKF-525A (27). Eth-
ylxanthate was found to inhibit CCl4 and pentobarbital
metabolism as well as to protect rats from nitrosodiethyl-
amine-induced liver carcinogenesis (28, 29).
In this study, n-propylxanthate (nPX, CH3CH2CH2-
OCS2) was found to inactivate the major phenobarbital
inducible rat liver P450 2B1 and the human 2B homo-
logue, P450 2B6, by a classical mechanism-based mech-
anism (30). The loss of enzymatic activity of P450 2B1
was primarily due to the binding of a nPX reactive
intermediate to the apoprotein.
En zym e Activity Assa ys a n d In a ctiva tion . Purified P450
2B1 and reductase were reconstituted with lipid for 1 h at 4
°C. Incubation mixtures contained 0.5 µM P450 2B1 or 0.67 µM
P450 2B6, 1 µM reductase, 200 µg/mL DLPC, 110 units/mL
catalase, and nPX or H2O in 50 mM potassium phosphate buffer
(pH 7.4). After equilibration of the reaction mixture at 30 °C
for 3 min, the reactions were initiated by adding NADPH to a
final concentration of 1.2 mM (primary reaction). The 7-EFC
O-deethylation activity was measured spectrofluorometrically
as described by Buters et al. (35). At the indicated times,
duplicate 10 µL samples (5 pmol of P450 2B1) of the primary
reaction mixture were removed and mixed with 990 µL of a
secondary reaction mixture containing 0.2 mM NADPH, 100 µM
7-EFC, and 40 µg/mL BSA in 50 mM potassium phosphate
buffer (pH 7.4), and incubated at 30 °C for 5 min. For P450 2B6,
duplicate 12 µL samples (8 pmol of P450 2B6) of the primary
reaction mixture were mixed with 988 µL of the secondary
reaction mixture and incubated for 10 min at 30 °C. Enzyme
activity was stopped by adding ice-cold acetonitrile to a final
concentration of 25%. The fluorescence of the samples was
measured directly at room temperature on an SLM-Aminco
model SPF-500 C spectrofluorometer with excitation at 410 nm
and emission at 510 nm. Enzyme activity was calculated from
a standard curve using HFC.
Exp er im en ta l P r oced u r es
Ma ter ia ls. Dilauroyl-L-R-phosphatidylcholine (DLPC), NAD-
PH, and catalase were purchased form Sigma Chemical Co. (St.
Louis, MO). 7-Ethoxy-4-(trifluoromethyl)coumarin (7-EFC) was
obtained from Molecular Probes, Inc. (Eugene, OR), and 7-hy-
droxy-4-(trifluoromethyl)coumarin (HFC) was from Enzyme
Systems Products (Livermore, CA). HPLC-grade methanol and
ethyl acetate were purchased from Mallinckrodt (Chesterfield,
MO). BCA reagent and Slide-A-Lyzer cassettes were from Pierce
(Rockford, IL). Ultima Gold liquid scintillation cocktail was
obtained from Packard (Meridien, CT). Centricon-30 micro
concentrators were obtained from Amicon (Danver, MA). [14C]-
Propanol was purchased from American Radionuclear (St. Louis,
MO). n-Propylxanthate and [14C]-n-propylxanthate were syn-
thesized as previously described (specific activity of 0.4 mCi/
mmol, radiochemical purity of 80%) (31). Purified P450 2B6 was
a kind gift from I. Hanna (Vanderbilt University, Nashville, TN).
P u r ifica tion of P 450 a n d Red u cta se. P450 2B1 and
reductase were purified from microsomes isolated from livers
of fasted male Long Evans rats (175-190 g, Harlan Sprague-
Dawley, Indianapolis, IN) given 0.1% phenobarbital in their
drinking water for 12 days according to the proceedure of Saito
and Strobel (32) and Strobel and Dignam (33), respectively.
Alternatively, reductase was purified after expression in Es-
cherichia coli as previously described (34). P450 2B6 was
expressed in E. coli MV1304 cells.2 Briefly, cultures were grown
in TB-peptone medium containing 17 mM KH2PO4, 72 mM K2-
HPO4, 12 g of tryptone, 24 g of yeast extract, 2 g of peptone, 4
mL of glycerol, and 100 mg/L ampicillin. Expression was induced
with 1 mM isopropyl â-O-thiogalactopyranoside, and the cul-
tures were grown at 130 rpm in the above medium supple-
mented with 0.5 mM δ-aminolevulinic acid, 1 mM thiamine, and
1 mg/L chloramphenicol for 3 days at 25 °C. Cultures that
exhibited a CO-reduced spectrum were pooled, and the cell pellet
was collected by centrifugation. Cells from 4 L were suspended
in 75 mM Tris-acetate (pH 8.0), 0.25 M sucrose, 0.25 mM EDTA,
and 12 mg of lysozyme in a final volume of 400 mL, and the
mixture was rocked on ice for 60 min. Spheroblasts were
collected by centrifugation and resuspended in 200 mL of 50
mM Tris-acetate (pH 8.0), 6 mM magnesium acetate, 0.5 M
NaCl, 10 mM â-mercaptoethanol, and 20% glycerol. Cells were
lysed by sonication, and the membranes were collected by
centrifuging the lysate for 1 h at 4 °C at 90000g. The membranes
were suspended in 200 mL of 50 mM Tris-acetate (pH 8.0), 0.5
M NaCl, 1% Triton N101 (v/v), 0.5% cholate (w/v), 10 mM
â-mercaptoethanol, and 40% glycerol (v/v) (solubilization buffer)
and solubilized for 5 h at 4 °C. The clarified supernatant was
adjusted to 5 mM with imidazole and applied to a Ni-NTA
agarose column (Qiagen, Valencia, CA) equilibrated with solu-
bilization buffer containing 5 mM imidazole. The column was
Su bstr a te P r otection . Substrate protection from nPX-
dependent inactivation of P450 2B1 was assayed by including
25 µM nPX together with 7-EFC at molar ratios of 1:0, 1:1, and
1:2 in the primary reaction mixture. At the indicated times,
duplicate 10 µL aliquots were removed and assayed for remain-
ing activity as described above.
P ar tition Ratio. To estimate the partition coefficient, samples
were incubated in the presence of 0.01-1 mM nPX for 20 min
to ensure the assay had proceeded to completion. Duplicate
aliquots were removed and assayed for 7-EFC activity as
described above.
Ir r ever sibility of In a ctiva tion of P 450 2B1 by n P X.
Cytochrome P450 2B1 (0.5 nmol) was reconstituted and inac-
tivated with 1.4 mM nPX in a total volume of 138 µL as
described above. Control samples were incubated with nPX but
without NADPH. After 10 min at 30 °C, the samples (0.13 mL)
were dialyzed overnight at 4 °C against 2 × 500 mL of 50 mM
potassium phosphate buffer (pH 7.4) containing 20% glycerol
and 0.1 mM EDTA. The dialyzed samples were reconstituted
with 10 µg of lipid for 30 min on ice. Some samples also received
fresh reductase. Enzymatic activity was assayed with 7-EFC
as described above.
Stoich iom etr y a n d Sp ecificity of Bin d in g. The stoichi-
ometry of binding was determined by extensively dialyzing 500
µL samples containing 2 nmol of P450 2B1 reconstituted with
reductase and lipid as described above that had been incubated
with 0.4 mM radiolabeled [14C]nPX, with or without NADPH
for 10 min at 30 °C. Aliquots were removed to assess the extent
of inactivation of P450 2B1 based on the residual 7-EFC
O-deethylation activity and to determine the amount of heme
loss by reduced CO difference spectroscopy prior to dialysis (36).
Samples were dialyzed in Slide-A-Lyzer cassettes against 4 ×
500 mL of 50 mM potassium phosphate (pH 7.4) containing 20%
glycerol, 10 mM sodium cholate, and 0.1 mM EDTA. Aliquots
were removed, and the amount of radioactivity remaining after
dialysis was measured by liquid scintillation counting. Cyto-
chrome P450 2B1 recovery was determined spectrophotometri-
cally by measuring the reduced CO difference spectra, and
protein recovery was quantified using the BCA reagent (37).
The stoichiometry of binding was calculated after subtracting
1
Abbreviations: reductase, NADPH-cytochrome P450 reductase;
DLPC, dilauroyl-L-R-phosphatidylcholine; nPX, n-propylxanthate; BSA,
bovine serum albumin; 7-EFC, 7-ethoxy-4-(trifluoromethyl)coumarin;
HFC, 7-hydroxy-4-(trifluoromethyl)coumarin; AHH-1, AHH-1 human
lymphoblastoid cell line.
2
I. Hanna et al., manuscript in preparation.

Inactivation of P450 2B1 and 2B6 by Propylxanthate
Chem. Res. Toxicol., Vol. 12, No. 4, 1999 319
Ta ble 1. Effect of n P X on th e 7-EF C O-Deeth yla tion
Activity of P u r ified P 450 2B1 a n d Cytoch r om e P 450
Con ten ta
% activity remaining % P450 remaining
primary reaction
0 min
20 min
0 min
20 min
P450 2B1
- nPX + NADPH
+ nPX - NADPH
+ nPX + NADPH
100
92 ( 1
91 ( 1
82 ( 4
80 ( 6
24 ( 2
100
88 ( 6
88 ( 2
75 ( 2
% activity remaining % P450 remaining
primary reaction
P450 2B6
0 min
30 min
0 min
30 min
- nPX + NADPH 100
+ nPX - NADPH 103 ( 2
+ nPX + NADPH
94 ( 2
102 ( 2
17 ( 1
100
100
102 ( 2
88 ( 1
F igu r e 1. Time- and concentration-dependent inactivation of
the P450 2B1 7-EFC O-deethylation activity by nPX. Aliquots
were removed from the primary reaction mixture and assayed
for residual activity as described in Experimental Procedures.
Each point shown represents the mean and standard deviation
from three separate experiments. For some points, the standard
deviation was smaller than the size of the symbol. The concen-
trations of nPX were (0) 0, (2) 20, (4) 25, (O) 30, ([) 35, and
(b) 50 µM. The inset shows the double-reciprocal plot of the rates
of inactivation as a function of nPX concentration.
a
Assay conditions were as described in Experimental Proce-
dures. The values shown represent the averages from two separate
experiments.
the background counts from dialyzed samples incubated with
nPX in the absence of NADPH.
Sp ectr op h otom etr ic Qu a n tita tion of P 450 2B1 a n d P 450
2B6. At the times indicated, 200 µL aliquots of the primary
reaction incubation were removed and diluted with 800 µL of
ice-cold 50 mM potassium phosphate (pH 7.4), containing 40%
glycerol and 0.6% Tergitol NP-40. The sample was gently
bubbled with CO for 90 s, and the spectrum was recorded from
400 to 500 nm on a DW2 UV/Vis spectrophotometer (SLM
Aminco, Urbana, IL) equipped with an OLIS spectroscopy
operating system (On-Line Instrument Systems, Inc., Bogart,
GA). Dithionite was added, and the reduced carbonyl spectrum
was recorded (36). For absolute spectral determinations, P450
2B1 and reductase were reconstituted with lipid at a 1:1 ratio.
The final concentrations were 1 µM P450 2B1, 1 µM reductase,
200 µg/mL DLPC, and 110 units/mL catalase in 50 mM
potassium phosphate (pH 7.4). The reference contained catalase
and lipid in 50 mM potassium phosphate (pH 7.4). Spectra were
recorded by scanning from 375 to 500 nm.
Iod osoben zen e-Su p p or ted Activity. P450 2B1 and reduc-
tase were reconstituted as described for the 7-EFC activity
assays above and incubated with 50 µM nPX and 0.5 mM
NADPH for 10 min at 30 °C. Aliquots were removed and diluted
into 990 µL of a secondary reaction mixture containing 100 µM
7-EFC and 50 mM potassium phosphate (pH 7.4). Either
NADPH at a final concentration of 0.2 mM or iodosobenzene at
a final concentration of 0.8 mM was added, and the fluorescence
emission at 510 nm was recorded over the course of 99 s. The
initial linear slope between 0 and 10 s was used to calculate
the percent activity remaining of the nPX-inactivated samples
as compared to control samples incubated with vehicle alone.
F igu r e 2. Time- and concentration-dependent inactivation of
the P450 2B6 7-EFC O-deethylation activity by nPX. Aliquots
were removed from the primary reaction mixture and assayed
for residual activity as described in Experimental Procedures.
The data shown represent the mean and standard deviation
from duplicate samples of three separate experiments. For some
points, the standard deviation was smaller than the size of the
symbol. The concentrations of nPX were (0) 0, (2) 8, (4) 10, ([)
20, (]) 30, (b) 40, and (O) 50 µM. The inset shows the double-
reciprocal plot of the rates of inactivation as a function of nPX
concentration.
Resu lts
incubated either without nPX or without NADPH did not
show reduced activity or diminished CO binding spectra.
The inactivation of P450 2B1 by nPX was time- and
concentration-dependent (Figure 1). Pseudo-first-order
inactivation kinetics were observed at 30 °C with nPX
concentrations ranging from 20 to 50 µM. The kinetic
constants describing the inactivation of P450 2B1 with
nPX were determined from the inset of Figure 1. The
maximal rate of inactivation at saturation (kinactivation) was
0.2 min-1; the concentration required for half-maximal
inactivation (KI) was 44 µM, and the time required for
half of the enzyme to become inactivated was 4.1 min.
No effect on the rates of inactivation was observed when
10 mM GSH or 1 mM DTT was added to the primary
incubation mixtures (data not shown).
In a ctiva tion of P 450 2B1 by n P X. After incubation
of reconstituted P450 2B1 with 50 µM nPX and NADPH
for 20 min, only 24% of the enzyme’s 7-EFC O-deethyl-
ation activity remained (Table 1). However, these samples
retained most of their ability to form a reduced CO
complex. Control incubations without nPX or with nPX
but without NADPH did not lead to a substantial loss in
either enzymatic activity or the ability of P450 2B1 to
bind CO. Similarly, 83% of the absolute absorbance
spectrum at 417 nm was retained after incubating for
20 min with nPX and NADPH (data not shown). When
P450 2B6 was incubated with 50 µM nPX and NADPH
for 30 min, an 83% loss in the 7-EFC O-deethylation
activity was observed. With the same samples, 88% of
the reduced CO complex was formed compared to control
samples incubated without nPX. Samples that were
Figure 2 shows that the inactivation of P450 2B6 by
nPX was also time- and concentration-dependent (Figure

320 Chem. Res. Toxicol., Vol. 12, No. 4, 1999
Kent et al.
F igu r e 4. Substrate protection against P450 2B1 inactivation
by nPX. Samples were removed from the primary incubation
mixture at the indicated time points and assayed for remaining
activity as described in Experimental Procedures. The data
shown are representative of two separate experiments. The
primary reaction mixtures contained molar rations of nPX to
7-EFC of (0) 0:0, ([) 1:0, (O) 1:1, and (2) 1:2.
F igu r e 3. Loss of P450 2B1 activity as a function of the ratio
of nPX to 2B1. P450 2B1 was incubated with different concen-
trations of nPX as described in Experimental Procedures. The
extrapolated partition ratio was determined from the intercept
of the linear regression line from the lower ratios and the
straight line obtained from the higher ratios.
Ta ble 2. Ir r ever sibility of P 450 2B1 In a ctiva tion by n P Xa
Ta ble 3. Iod osoben zen e-Su p p or ted Activity of
n P X-In a ctiva ted P 450 2B1a
% activity remaining
sample
before dialysis
after dialysis
after dialysis with fresh reductase
control
inactivated
% activity
100
100
100
10 ( 2
11 ( 1
14 ( 1
compd supporting activity
control
100
105 ( 15
inactivated
NADPH
iodosobenzene
7.8 ( 0.2
59 ( 10
a
Assay conditions were as described in Experimental Proce-
dures. The data shown represent the average of two separate
experiments.
a
Assay conditions were as described in Experimental Proce-
dures. The data shown represent the average of two separate
experiments.
2). The kinetic constants were determined from the inset
of Figure 2. The KI was 12 µM; the kinactivation was 0.06
min-1, and the t1/2 was 11 min.
Stoich iom etr y of La belin g. The stoichiometry of
binding of a radiolabeled nPX metabolite to P250 2B1
was determined. Control samples incubated only with
[14C]nPX and inactivated samples incubated with [14C]-
nPX and NADPH were dialyzed extensively until no more
counts were detected in the dialysis buffer. Sample
aliquots were assayed for P450 recovery by reduced CO
spectroscopy and for radioactivity by liquid scintillation
counting. Background counts from control samples in-
cubated without NADPH (344 and 471 dpm/nmol of P450
2B1) were subtracted from the counts obtained for the
inactivated samples (1124 and 1353 dpm/nmol of P450
2B1). Determinations from two separate experiments
resulted in an average binding stoichiometry of 1.2 mol
of metabolite bound per mole of inactivated P450 2B1.
Iod osoben zen e-Su p p or ted Activity. The alternate
oxidant iodosobenzene was used to test if substrate
binding by the nPX-treated P450 2B1 samples was
compromised. In all cases, the rates of 7-EFC metabolism
supported by iodosobenzene were linear only for the first
10-15 s. The rates observed during the first 10 s were
used to calculate the data shown in Table 3. When
NADPH was used to support the 7-EFC metabolism, only
7% of the O-deethylation activity in samples that had
been inactivated with nPX was seen compared to control
samples. When iodosobenzene was used as the alternate
oxidant, 59% of the control O-deethylation activity was
observed.
P a r tition Ra tio. The number of molecules of nPX
metabolized per molecule of 2B1 inactivated (partition
ratio) was estimated from Figure 3. P450 2B1 was
incubated with different concentrations of nPX, and the
inactivation reaction was allowed to go to completion. The
percent activity remaining was plotted as a function of
the molar ratio of nPX to 2B1. The turnover number
(partition ratio + 1) was extrapolated from the intercept
between the linear regression line obtained from the
lower nPX to 2B1 ratios with the straight line derived
from the higher nPX to P450 2B1 ratios (30). With this
method, the partition ratio was found to be 32.
Ir r ever sibility of 2B1 In a ctiva tion by n P X. Control
samples or samples where P450 2B1 was inactivated with
nPX were dialyzed extensively and tested for activity.
Table 2 shows that the removal of free nPX by dialysis
did not lead to a recovery of the 7-EFC activity of nPX-
inactivated samples. No additional activity was regained
when fresh reductase was added to the dialyzed samples.
Su bstr a te P r otection . Incubations of P450 2B1 with
nPX together with an alternate substrate in the primary
reaction mixture slowed the rate at which nPX inacti-
vated P450 2B1 (Figure 4). Virtually no inactivation was
seen when the alternate substrate concentration was
twice that of nPX.

Inactivation of P450 2B1 and 2B6 by Propylxanthate
Chem. Res. Toxicol., Vol. 12, No. 4, 1999 321
binding occurred at the active site and that approxi-
mately one molecule of inactivator was bound per mol-
ecule of P450 2B1.
Discu ssion
This report describes the mechanism-based inactiva-
tion of rat P450 2B1 and human P450 2B6 by n-
propylxanthate. nPX was chosen for these studies be-
cause it is structurally distinct from the mechanism-
based inactivators that have previously been used to label
the active site of P450 2B1 (10-12). While both isoforms
were inactivated with nPX, little effect on either the P450
2B1 or P450 2B6 reduced CO spectrum was observed.
The data in Table 1 suggested that the inactivation by
nPX was not due to the destruction of the heme moiety
but rather to a modification of the apoprotein. Additional
evidence that the heme moiety was not affected came
from analysis of butanone extracts of P450 2B1 that was
inactivated with 14C-labeled nPX. No counts were ob-
served in the heme-containing fraction of these samples
(data not shown). In addition, our data show that
inactivation by nPX required a catalytic step since both
NADPH and nPX were necessary to inactivate P450 2B1
and P450 2B6.
Pseudo-first-order kinetics for inactivation were ob-
served with both enzymes. Although the two P450s share
approximately 76% sequence similarity (38), a 4-fold
difference between the KI of P450 2B1 (44 µM) and that
of P450 2B6 (12 µM) for nPX was observed. The rate of
inactivation for P450 2B6 was slower (0.06 min-1) when
compared to the kinactivation for P450 2B1 (0.2 min-1).
Similar differences between the two enzymes in the KI
and kinactivation were seen when AHH-1 microsomes ex-
pressing P450 2B6 were compared to P450 2B13 and
when the kinetics of inactivation by 2-phenyl-2-piperi-
dinopropane of the purified P450 2B1 and P450 2B6 were
compared.4
P450 2B1 samples that were incubated with nPX or
inactivated with nPX and NADPH were dialyzed exten-
sively to determine if removal of free inactivator would
restore enzymatic activity, or if the inactivation was
reversible. No regain of enzymatic activity of the inacti-
vated sample compared to the control sample was ob-
served after dialysis. These results suggest that under
these conditions the inactivation was irreversible. The
inactivation appeared to be due to a modification of the
P450 and not the reductase since adding fresh reductase
back to the dialyzed samples also did not restore enzy-
matic activity to the nPX-inactivated P450 2B1. In
addition, no loss in enzymatic activity was observed when
P450 2B1 was incubated with nPX together with a 2-fold
molar excess of an alternate substrate. This observation
suggested that nPX inactivated P450 2B1 and that the
inactivation was due to binding of a nPX reactive
intermediate to the P450 active site. The addition of
exogenous nucleophiles such as glutathione (1 or 10 mM)
or dithiothreitol (1 mM) to the reaction mixture had no
effect on the rates of inactivation (data not shown). These
observations further indicate that the inactivation of
P450 2B1 by nPX was due to the binding of a nPX
reactive intermediate at the active site and not because
the intermediate diffused out of the active site and bound
elsewhere on the P450 molecule or the reductase. In
addition, the 1.2:1 stoichiometry of radiolabeled nPX
metabolite to P450 2B1 would also suggest that the
Significant enzymatic activity could be restored to nPX-
inactivated samples when iodosobenzene was used as an
alternate oxidant in place of NADPH and O2. This
observation suggests that the nPX reactive intermediate
bound to an amino acid residue(s) critical for a reductase-
dependent step. Alternatively, the modification may have
altered the binding or dissociation of the substrate and
favored oxidation with iodosobenzene. A third possibility
is that the nPX modification disrupted a proton transfer
step required to generate the ferryl intermediate. Experi-
ments designed to isolate the nPX-labeled peptide and
to identify the modified amino acid(s) are in progress.
The reactive intermediate responsible for inactivating
P450 2B1 and P450 2B6 has not been identified. Earlier
reports from in vivo experiments where rats were injected
with radiolabeled xanthates showed that the correspond-
ing alkanes and alkenes appeared in the animal’s expired
air (27). These observations led the investigators to
propose a possible reaction mechanism that involved an
attack at the R-carbon to generate an alkane radical that,
in the presence of H+ or OH-, could be converted to the
alkane or alkene, respectively (27). With the purified
enzyme, no corresponding alkane or alkene could be
detected in the headspace by gas chromatography (data
not shown). However, propionaldehyde was able to
inactivate P450 2B1 at millimolar concentrations but
could not compete for labeling of the enzyme by nPX.
3
When nPX that was H-labeled at the R-carbon was used
to inactivate P450 2B1, all the counts were released into
the buffer (data not shown). When nPX labeled with 14C
at the R-carbon was used, the counts remained with the
P450 protein (data not shown). Taken together, these
observations suggest that the initial site of oxidation by
P450 2B1 is the R-carbon of nPX and that the inactivat-
ing species could be a hydroxylated propyl radical.
Experiments designed to test this hypothesis are in
progress.
In summary, this report demonstrates that nPX is an
effective mechanism-based inactivator for both rat P450
2B1 and human P450 2B6. The data indicate that the
reactive intermediate generated during the metabolism
of nPX bound to the P450 2B1 active site. Experiments
designed to identify the amino acid residue(s) involved
in the inactivation are in progress.
Ack n ow led gm en t. This publication was supported
in part by NIH Grant CA 16954 (P.F.H.) from the
National Cancer Institute. Its contents are solely the
responsibility of the authors and do not necessarily
represent the official views of the National Cancer
Institute. We thank Dr. Frank Gonzalez for his kind gift
of P450 2B6 plasmid. We are particularly grateful to Drs.
Alfin Vaz and Dermot McGinnity for their help with the
gas chromatographic analysis and to Dr. Vaz for the
many helpful discussions.
Refer en ces
(1) Porter, T. D., and Coon, M. J . (1991) Cytochrome P450. Multiplic-
ity of isoforms, substrates, and catalytic and regulatory mecha-
nisms. J . Biol. Chem. 266, 13469-13472.
(2) Gonzalez, F. J . (1988) The molecular biology of cytochrome P450s.
Pharm. Rev. 40, 243-288.
3
S. Yanev, U. M. Kent, B. Pandova, and P. F. Hollenberg, manu-
script in preparation.
4
(3) Nelson, D. R., Koymans, L., Kamataki, T., Stegeman, J . J .,
Feyereisen, R., Waxman, D. J ., Waterman, M. R., Gotoh, O., Coon,
J . Chun, U. M. Kent, R. Moss, L. Sayre, and P. F. Hollenberg,
manuscript in preparation.

322 Chem. Res. Toxicol., Vol. 12, No. 4, 1999
Kent et al.
M. J ., Estabrook, R. W., Gunsalus, C., and Nebert, D. W. (1996)
P450 superfamily: update on new sequences, gene mapping,
accession numbers and nomenclature. Pharmacogenetics 6, 1-42.
(4) Halpert, J . R. (1995) Structural basis of selective cytochrome P450
inhibition. Annu. Rev. Pharmacol. Toxicol. 35, 29-53.
(5) Kedzie, K. M., Baltour, C. A., Escobar, G. Y., Grimm, S. W., He,
Y.-A., Pepperl, D. J ., Regan, J . W., Stevens, J . C., and Halpert, J .
R. (1991) Molecular basis for a functionally unique cytochrome
P450IIBI variant. J . Biol. Chem. 266, 22515-22521.
(6) Poulos, T. L., Finzel, B. C., and Howard, A. J . (1987) High-
resolution crystal structure of cytochrome P450 cam. J . Mol. Biol.
195, 687-700.
(7) Ravichandran, K. G., Boddupalli, S. S., Hasemann, C. A., Peter-
son, J . A., and Deisenhofer, J . (1993) Crystal structure of
hemoprotein domain of P450 BM-3, a prototype for microsomal
P450s. Science 261, 731-736.
(8) Hasemann, C. A., Ravichandran, K. G., Peterson, J . A., and
Deisenhofer, J . (1994) Crystal structure and refinement of cyto-
chrome P450terp at 2.3 Å resolution. J . Mol. Biol. 236, 1169-
1185.
(9) Cupp-Vickery, J . R., and Poulos, T. L. (1995) Structure of
cytochrome P450eryF involved in erythromycin biosynthesis. Nat.
Struct. Biol. 2, 144-153.
(10) Roberts, E. S., Hopkins, N. E., Alworth, W. L., and Hollenberg,
P. F. (1993) Mechanism-based inactivation of cytochrome P450
2B1 by 2-ethynylnaphthalene: Identification of an active site
peptide. Chem. Res. Toxicol. 6, 470-479.
(11) Roberts, E. S., Hopkins, N. E., Zaluzec, E. J ., Gage, D. A., Alworth,
W. L., and Hollenberg, P. F. (1995) Mechanism-based inactivation
of cytochrome P450 2B1 by 9-ethynylphenathrene. Arch. Biochem.
Biophys. 323, 295-302.
(12) He, K., Falick, A. M., Chen, B., Nilsson, F., and Correia, M. A.
(1996) Identification of the heme adduct and an active site peptide
modified during mechanism-based inactivation of rat liver cyto-
chrome P450 2B1 by secobarbital. Chem. Res. Toxicol. 9, 614-
622.
Genotoxicity of tamoxifen, tamoxifen epoxide and tormifene in
human lymphoblastoid cells containing human cytochrome P450s.
Carcinogenesis 15, 5-9.
(22) Ono, S., Hatanaka, T., Hotta, H., Satoh, T., Gonzalez, F. J ., and
Tsutsui, M. (1996) Specificity of substrate and inhibitor probes
for cytochrome P450s: evaluation of in vitro metabolism using
cDNA expressed human P450s and human liver microsomes.
Xenobiotica 26, 681-693.
(23) Yamazaki, H., Inoue, K., Mimura, M., Oda, Y., Guengerich, F.
P., and Shimada, T. (1996) 7-Ethoxycoumarin O-deethylation
catalyzed by cytochromes P4501A2 and 2E1 in human liver
microsomes. Biochem. Pharmacol. 51, 313-319.
(24) Imaoka, S., Yamada, T., Hiroi, T., Hayashi, K., Sakaki, T.,
Yabusaki, Y., and Funae, Y. (1996) Multiple forms of human P450
expressed in Saccharomyces cerevisiae. Biochem. Pharmacol. 51,
1041-1050.
(25) Stevens, J . C., White, R. B., Hsu, S. H., and Martinet, M. (1997)
Human liver CYP2B6-catalyzed hydroxylation of RP 73401. J .
Pharmacol. Exp. Ther. 282, 1389-1395.
(26) Roberts, E. S., Hopkins, N. E., Foroozesh, M., Alworth, W. L.,
Halpert, J . R., and Hollenberg, P. F. (1997) Inactivation of
cytochrome P450s 2B1, 2B4, 2B6, and 2B11 by arylalkynes. Drug
Metab. Dispos. 25, 1242-1248.
(27) Yanev, S., Frank, H., Remmer, H., and Stoytchev, T. (1985)
Xanthogenates as “suicide” substrates of cyt-P-450. In Cytochrome
P450, Biochemistry, Biophysics and Induction (Vereczkey, L., and
Magyar, K., Eds.) pp 87-90, Elsevier, New York.
(28) Yanev, S., Yanku, I., Stoychev, T., Havilk, I., and Krebs, V. (1982)
Effects of potassium ethylxanthogenate and 2,3-dimercapto-
propane sulphonate sodium on the pentobarbital pharmacoki-
netics and metabolism in male mice. Eur. J . Drug Metabol.
Pharmacokinet. 7, 21-29.
(29) Hadjiolov, D., Frank, M., Hadjiolov, N., and Yanev, S. (1988)
Effects of potassium ethylxanthogenate on nitrosodiethyl-
amine-induced DNA damage and on liver carcinogenesis. Arch.
Geschwulstforsch. 58, 313-318.
(30) Silverman, R. B. (1996) Mechanism-based enzyme inactivators.
In Contemporary Enzyme Kinetics and Mechanisms (Purich, D.
L., Ed.) pp 291-335, Academic Press, San Diego, CA.
(31) Rao, S. R. (1971) Xanthates and related compounds, pp 7-31,
Marcel Dekker, New York.
(32) Saito, T., and Strobel, H. W. (1981) Purification to homogeneity
and characterization of a form of cytochrome P450 with high
specificity for benzopyrene from â-naphthoflavone-pretreated rat
liver microsomes. J . Biol. Chem. 256, 984-988.
(33) Strobel, H. W., and Dignam, J . D. (1978) Purification and
properties of NADPH-cytochrome P-450 reductase. Methods En-
zymol. 52, 89-92.
(34) Hanna, I. H., Teiber, J . F., Kokones, K. L., and Hollenberg, P. F.
(1998) Role of alanine at position 363 of cytochrome P450 2B2 in
influencing the NADPH- and hydroperoxide-supported activities.
Arch. Biochem. Biophys. 350, 324-332.
(13) Rylander, T. M., Hellmold, H., Reihne´r, E., Warner, M., and
Gustafsson J .-Å. (1997) RT-PCR analysis of cytochrome P450
profile in normal and tumor samples from human breast. FASEB
J . 11, P343.
(14) Osborne, M. P., Bradlow, H. L., Wong, G. Y., and Telang, T. N.
(1993) Upregulation of estradiol C16 alpha-hydroxylation in
human breast tissue: a potential biomarker of breast cancer risk.
J . Natl. Cancer Inst. 85, 1917-1920.
(15) Ekins, S., VandenBranden, M., Ring, B. J ., and Wrighton, S. A.
(1997) Examination of purported probes of human CYP 2B6.
Pharmacogenetics 7, 165-179.
(16) Shimada, T., Yamazki, H., Mimura, M., Inui, Y., and Guengerich,
F. P. (1994) Interindividual variation in human liver cytochrome
P-450 enzymes involved in the oxidation of drugs, carcinogens
and toxic chemicals: Studies with liver microsomes of 30 J apa-
nese and 30 Caucasians. J . Pharmacol. Exp. Ther. 270, 414-423.
(17) Madan, A., LeCluyse, E. L., Foster, J ., Carroll, K., Lantham, J .
D., Yang, D., and Parkinson, A. (1996) Identification and char-
acterization of cytochrome P450 2B6 (CYP2B6) in human liver
microsomes and its induction in primary cultures of human
hepatocytes. XI International Symposium on Microsomes and
Drug Oxidations, UCLA, Los Angeles, CA, J uly 21-24, 1996, p
203.
(35) Buters, J . T., Schiller, C. D., and Chou, R. C. (1993) A highly
sensitive tool for the assay of cytochrome P450 enzyme activity
in rat, dog and man. Biochem. Pharmacol. 46, 1577-1584.
(36) Omura, T., and Saito, R. (1964) The carbon monoxide-binding
pigment of liver microsomes. I. Evidence for its hemo protein
nature. J . Biol. Chem. 239, 2370-2378.
(18) Yang, T. J ., Krausz, K. W., Shou, M., Yang, S. K., Buters, J . T.,
Gonzalez, F. J ., and Gelboin, H. V. (1998) Inhibitory monoclonal
antibody to human cytochrome P450 2B6. Biochem. Pharmacol.
55, 1633-1640.
(19) McCracken, N. W., Cholerton, S., and Idle, J . R. (1992) Cotinine
formation by cDNA-expressed human cytochromes P450. Med.
Sci. Res. 20, 877-878.
(20) Mimura, M., Baba, T., Yamazaki, H., Ohmori, S., Inui, Y.,
Gonzalez, F. J ., Guengerich, F. P., and Shimada, T. (1993)
Characterization of cytochrome P-450 2B6 in human liver mi-
crosomes. Drug Metab. Dispos. 21, 1048-1056.
(21) Styles, J . A., Davies, A., Lim, C. K., DeMatteis, F., Stanley, L.
A., White, I. N. H., Yuan, Z.-X., and Smith, L. L. (1994)
(37) Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K.,
Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N.
M., Olson, B. J ., and Klenk, D. C. (1985) Measurement of protein
using bicinchoninic acid. Anal. Biochem. 150, 76-85.
(38) Yamano, S., Nhamburo, P. T., Aoyama, T., Meyer, U. A., Inaba,
T., Kalow, W., Gelboin, H. V., McBride, O. W., and Gonzalez, F.
J . (1989) cDNA cloning and sequence and cDNA-directed expres-
sion of human P450 IIB1: identification of a normal and two
variant cDNAs derived from the CYP2B locus on chromosome 19
and differential expression of the IIB mRNAs in human liver.
Biochemistry 28, 7340-7348.
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