The mechanism of oxidation of allylic alcohols to α,β-unsaturated ketones by cytochrome P450 a

Article abstract of DOI:10.1021/tx9600053

The oxidation of cyclohex-2-en-1-ol, a simple model substrate for allylic alcohols, is catalyzed by several P450 isoenzymes and leads exclusively to cyclohex-2-en-1-one. No double bond epoxidation or C(4) hydroxylation have been observed. The large primary kinetic isotope effect measured using [²H]-1-cyclohex-2-en-1-ol is consistent with an at least partially rate limiting breaking of the C(1)-H bond. The mass spectrometric analysis of cyclohex-2-en-1-one obtained from [¹⁸O]cyclohex-2-en-1-ol has established that a gem-diol intermediate is involved, even if a dual hydrogen abstraction pathway may contribute to the reaction.

Full text of DOI:10.1021/tx9600053

Chem. Res. Toxicol. 1996, 9, 871-874
871
Th e Mech a n ism of Oxid a tion of Allylic Alcoh ols to
r,â-Un sa tu r a ted Keton es by Cytoch r om e P 450†
Giuseppe Bellucci,‡ Cinzia Chiappe,*,‡ Laura Pucci,§ and
Pier Giovanni Gervasi*,§
Dipartimento di Chimica Bioorganica, Universita` di Pisa, via Bonanno 33, 56126 Pisa, Italy, and
Istituto di Mutagenesi e Differenziamento, CNR, via Svezia 10, 56124 Pisa, Italy
Received J anuary 10, 1996X
The oxidation of cyclohex-2-en-1-ol, a simple model substrate for allylic alcohols, is catalyzed
by several P450 isoenzymes and leads exclusively to cyclohex-2-en-1-one. No double bond
epoxidation or C(4) hydroxylation have been observed. The large primary kinetic isotope effect
measured using [2H]-1-cyclohex-2-en-1-ol is consistent with an at least partially rate limiting
breaking of the C(1)-H bond. The mass spectrometric analysis of cyclohex-2-en-1-one obtained
from [18O]cyclohex-2-en-1-ol has established that a gem-diol intermediate is involved, even if
a dual hydrogen abstraction pathway may contribute to the reaction.
In tr od u ction
Ma ter ia ls a n d Meth od s
Ch em ica ls. Dexamethasone (DEX), â-naphtoflavone (â-NF),
phenobarbital (PB), and pyrazole (PZ) were obtained from
common commercial sources. Cyclohex-2-en-1-ol (1) was pre-
pared by refluxing 3-bromocyclohexene (2 g) in 7.5 mL of
tetrahydrofuran-water (2:1) containing Na2CO3 (1.4 g) for 4 h,
Cytochrome P450s are a family of isoenzymes able to
oxidize a broad variety of endogenous and exogenous
substrates playing a crucial role in the metabolism of
xenobiotics (1). Olefinic compounds are usually oxidized
by cytochrome P450s to the corresponding epoxides (2),
while alcoholic functions can be converted to carbonyl
groups (3). The latter biotransformation is particularly
prominent for allylic alcohols, where double bond epoxi-
dation appears to be hampered in favor of alcoholic group
oxidation. This is particularly important from the toxi-
cological point of view, since epoxides are electrophilically
reactive compounds able to react with the cellular
macromolecules producing toxic, mutagenic, and carci-
nogenic effects (4). A typical example of allylic alcohol
oxidation is provided by the metabolism of trans-R,R,4-
trimethyl-5-hydroxy-3-cyclohexene-1-methanol (sobrerol),
a mucoregulatory drug, whose main metabolite is the
corresponding R,â-unsaturated ketone (5). No double
bond epoxidation of sobrerol was detected, in spite of the
fact that 1-methylcyclohexene, a model compound for
sobrerol lacking the allylic hydroxyl group, was exten-
sively epoxidated by liver microsomal preparations from
control and induced rats (6).
followed by distillation (bp 164-165 °C).
[
18O]-1-Cyclohexen-
2-en-1-ol ([18O]-1) was prepared on a 10-fold reduced scale using
the same procedure, employing commercial (Aldrich) [18O]water,
followed by Kugelrohr short-path distillation. [2H]-1-Cyclohex-
2-en-1-ol ([2H]-1, >95% [2H], by NMR) was obtained by reduction
of commercial cyclohex-2-en-1-one (2) with an equimolar amount
of LiAlD4 in anhydrous ethyl ether at room temperature. cis-
2,3-Epoxycyclohexanol and trans-2,3-epoxycyclohexanol were
prepared by epoxidation of 1 with m-chloroperbenzoic acid in
dichloromethane. Cyclohex-2-ene-1,4-diol was prepared as
previously reported (7).
An im a ls a n d Micr osom a l P r ep a r a tion s. Male Sprague-
Dawley rats were purchased from Charles River, Germany.
They were treated for 3 days with PB ip (80 mg/kg daily), DEX
(40 mg/kg daily), â-NF (40 mg/kg daily), or PZ (200 mg/kg daily),
and microsomes were obtained from the liver as previously
described (8). Microsomal protein concentrations were assayed
by using the method of Lowry et al. (9); the total cytochrome
P450 concentration was measured according to Omura and Sato
(10).
En zym a tic Activity a n d Kin etic P a r a m eter s. 1-Cyclo-
hex-2-en-1-ol oxidase activity was usually measured as follows.
Incubation mixtures (2 mL), containing 100 mM K/phosphate
buffer, pH 7.4, 2 mg of hepatic microsomal protein, and an
NADPH-generating system, consisting of 0.5 mM NADP+, 5 mM
glucose 6-phosphate, and 0.5 unit/mL glucose 6-phosphate
dehydrogenase, were incubated for 5 min, and the reactions
were initiated by the addition of a proper amount of 1 or its
[2H]-1 or [18O] isotopomers. After 15 min, NaCl was added to
precipitate the microsomal proteins. The reaction products were
extracted with ethyl acetate (2 × 5 mL) and quantified by GLC
(OV 225 column, 95 °C) after addition of appropriate amounts
of an ethyl acetate stock solution of cyclohexanone as an internal
standard. The substrate concentrations ranged from 1 to 50
mM. Concentrations of 1 higher than 50 mM were not used as
they may cause microsomal enzyme denaturation. 1-Cyclohex-
2-en-1-ol oxidase activity was also measured in reconstituted
systems by using purified 2B1, 2E1, 1A1, and NADPH cyto-
chrome P450 reductase obtained as previously described (8, 11),
and 2C11 purified from control male SD rats according to Fugita
et al. (12).
In order to focus on the factors affecting the competi-
tion between double bond epoxidation and hydroxyl group
oxidation and to try to identify the involved P450s, as
well as to clarify the mechanistic aspects of this reaction,
we undertook a study of the oxidative biotransformation
of cyclohex-2-en-1-ol (1) by rat liver P450s. The use as
sources of P450s of microsomal preparations obtained
after pretreatment of rats with P450 selective inducers,
and the use of some purified P450s (1A1, 2E1, 2B1,
2C11), has shown that 1 is susceptible of oxidation by
some P450 isoforms only at its OH group. In addition,
2
the use of 18O or H labeled substrate has allowed us to
formulate a mechanistic rationalization of the investi-
gated reaction.
† This paper is dedicated to the memory of Professor Giuseppe
Bellucci (d. March 3, 1996).
‡ Universita` di Pisa.
§ Istituto di Mutagenesi e Differenziamento, CNR.
X Abstract published in Advance ACS Abstracts, J une 15, 1996.
The reconstituted incubation mixtures (0.5 mL) contained 0.2
nmol of P450, 0.6 nmol of P450 reductase, 15 µg of dilau-
S0893-228x(96)00005-7 CCC: $12.00 © 1996 American Chemical Society

872 Chem. Res. Toxicol., Vol. 9, No. 5, 1996
Bellucci et al.
Ta ble 1. Va lu es of Ap p a r en t Kin etic Con sta n ts for th e
Oxid a tion of 1 by Micr osom es fr om Con tr ol a n d Tr ea ted
Ra ts a n d by P 450 2C11, 2B1, 2E1, a n d 1A1a
roylphosphatidylcholine, and 1 in 0.1 M K/phosphate buffer, pH
7.4. The reactions were carried out at 37 °C for 15 min after
the addition of 1 mM NADPH, and the products were extracted
and analyzed as described above.
microsomes or
purified P450
Km
(mM)
Vmax [nmol min-1 Vmax [nmol min-1
(mg of protein)-1
]
(nmol of P450)-1
]
In all cases, the oxidation of 1 to 2 followed simple Michaelis-
Menten kinetics. With control microsomes it was linear up to
20 min and 1 mg/mL of microsomal protein. The apparent
kinetic parameters Vmax and Km were determined from Lin-
eweaver-Burk plots of 2 formation data obtained from incuba-
tions containing eight different substrate concentrations.
Control experiments carried out incubating cis-2,3-epoxycy-
clohexanol, trans-2,3-epoxycyclohexanol, and cyclohex-4-ene-1,4-
diol for the same time as 1 showed that these compounds were
completely recovered.
control
â-NF
PZ
PB
DEX
79 ( 10
29 ( 3**
22 ( 8**
17 ( 4**
9 ( 4**
28 ( 5
33 ( 4
22 ( 3
17 ( 5
9 ( 3**
51 ( 9
27 ( 4*
32 ( 5*
19 ( 6**
11 ( 4**
P450 2C11
P450 2B1
P450 2E1
P450 1A1
72 ( 11
20 ( 5
NDb
55 ( 8
11 ( 3
3.2 ( 0.7c
2.1 ( 0.5c
NDb
Ma ss Sp ectr a l Deter m in a tion s. The ratio between the
[16O] and [18O] isotopomers of 1 was determined by GC-MS using
a Perkin-Elmer 8200 QMass 910 equipped with a DB1 column
(70 °C, retention time 4.46 min), on the basis of the M - 1, M,
and M + 1 peaks at m/z 97, 98, and 99 (for the unlabeled) and
at 99, 100 and 101 (for the [18O] labeled compound), as well as
on the basis of the M - 15 peaks at m/z 83 and 85. Both
determinations gave an 80:20 ratio of the labeled to the
unlabeled alcohol. The product of the incubation of a sample
of this alcohol (5 mg) with a microsomal preparation (5 mL)
obtained from PB pretreated animals was subjected to GC-MS
analysis under the above conditions (retention time of 2, 5.1
min). A 50:50 ratio of the [16O] and [18O] isotopomers was
determined on the basis of the molecular ion peaks at m/z 96
and 98. This isotopomer ratio remained exactly unchanged
when the oxidation product was left in the experimental
conditions for a much longer time, showing that oxygen ex-
change on the ketone with H2O did not occur at an appreciable
rate.
a
The reported values are averages ( SD of three determina-
b
tions performed with different microsomal preparations. ND )
not determined. c Value obtained at 20 mM concentration of 1. (**
and *) Significantly different from the control microsomes by
Student’s t-test: **P <0.01; *P <0.05.
production of 2 on the basis of P450 content (Table 1).
These findings suggested that none of the above isoen-
zymes is a more effective catalyst of the oxidation of 1
than those present in control microsomes.
This was confirmed by using the purified P450 1A1,
2E1, 2B1, and 2C11, the major constitutive P450 of rat
liver (13), in a reconstituted system. P450 2E1, 1A1, and
also 2B1 exhibited a much lower turnover compared to
P450 2C11. It should be noted, however, that P450 2C11
had a Km higher than that of 2B1, consistent with the
Km data found in control and PB-induced microsomes,
respectively.
It is also noteworthy that the ratios of Vmax/Km for 2B1
and 2C11 were not too dissimilar, being 0.55 and 0.76,
respectively. This may be indicative that both enzymes,
in proportion to their expressions in microsomes, play an
important role in the oxidation of 1 (14).
Com p a r ison of Micr osom a l Oxid a t ion of 1 a n d
[2H]-1. To check the nature of the rate limiting step of
the oxidation of the C(1) hydroxyl group, 1 and [2H]-1
were incubated with PB-induced microsomes and the
production of 2 was determined by GLC. PB-induced
Resu lts
Micr osom a l Oxid a tion of 1-Cycloh ex-2-en -1-ol (1).
The allylic alcohol 1 was oxidized by microsomal enzymes
in the presence of an NADPH-generating system only at
the hydroxyl group, giving the corresponding ketone 2.
Neither products of epoxidation of the double bond (cis-
and trans-2,3-epoxycyclohexanols or triols arising from
their hydrolysis) nor products of allylic hydroxylation at
C(4) of 1 (cyclohex-2-ene-1,4-diol) were detected by GLC
of the extracts of the incubation mixtures. That these
products were actually not formed was confirmed by
control experiments showing that they were stable in the
incubation conditions.
microsomes were chosen because they gave the highest
amounts of 2 at the lowest 1 concentration. From linear
Lineweaver-Burk plots of the initial rates of these
oxidations, the following apparent Vmax and Km values
were obtained: 16 nmol min-1 (mg of protein)-1 and 18
mM for 1, and 2.9 nmol min-1 (mg of protein)-1 and 21
mM for [2H]-1, respectively. The data show that, whereas
the Km did not change, as expected, on substitution of a
hydrogen with a deuterium at C(1), the apparent Vmax
decreased by a factor of 5.5. This can be attributed to a
primary kinetic isotope effect on the at least partially rate
limiting breaking of the C(1)-L (L ) H or D) bond (15).
Oxid a tion of [18O]-1. To investigate the fate of the
intermediate arising from the rate determining abstrac-
tion of the C(1)-H bond, a sample of 80% 18O labeled
allylic alcohol, obtained by hydrolysis of 3-bromocyclo-
hexene with 18OH2, was incubated with PB-induced
microsomes. The formed ketone was analyzed by GC-
MS, which showed molecular ions at m/z 96 and 98 in a
50:50 ratio. This proved that an extensive scrambling
The formation of 2 followed monophasic Michaelis-
Menten kinetics and required NADPH, whereas NADH
was rather ineffective as a source of electrons. The
oxidation also required, as typical for P450-dependent
reactions, molecular oxygen and was inhibited by CO,
•
whereas SOD and mannitol, scavengers of O2•- and OH,
respectively, did not inhibit the formation of 2, indicating
that the above radicals are not involved in the oxidation
of 1.
Effect of P r etr ea tm en ts w ith P 450 In d u cer s a n d
Use of P u r ified P 450 1A1, 2B1, 2E1, a n d 2C11.
Pretreatments of rats with â-NF, PZ, PB, and DEX,
respective classical inducers of P450 1A1/2, 2E1, 2B1/2,
and 3A1/2 (13), significantly decreased, compared to the
control values, both the apparent Km and the Vmax for the

Oxidation of Allylic Alcohols by Cytochrome P450
Chem. Res. Toxicol., Vol. 9, No. 5, 1996 873
Sch em e 1
mechanism, if complete conformational equilibration
occurs in the gem-diol (see below), since 16OH2 and 18OH2
should be eliminated with the same probability. Alter-
natively, double hydrogen abstraction pathways may be
involved, consisting in the reaction of the perferric
hydroxide radical with the hydroxyl hydrogen (path b)
or with a hydrogen attached to a carbon adjacent to the
initial carbon radical to give the enolic form of the ketone
(path c). The last pathway has been observed (17) in the
cytochrome P450-catalyzed oxidation of ethyl carbamate
to vinyl carbamate. Complete retention of the [18O] label
must be observed for pathways b and c.
The mass spectrometric analysis of the starting [18O]
labeled alcohol and of the resulting ketone, after correc-
tion for the 20% unlabeled alcohol present in the sample
subjected to oxidation, shows that the [18O] and [16O]
isotopomers of 2 were produced from [18O]-1 in a 5:3 ratio
and that this ratio is not affected by oxygen exchange
with water. This shows that pathway a is certainly
operative. In this framework, the observed preference
for the 18O retention in the ketone could be due to
incomplete conformational equilibration of the gem-diol
before loss of water. It has been shown (18) that in the
cytochrome P450 2B1-catalyzed oxidation of testosterone
and epitestosterone to androstenedione in an 18O2 atmo-
sphere a gem-diol intermediate is formed, which under-
goes a stereoselective loss of water from the R-face. If 1
undergoes C-1 hydroxylation at the pseudoaxial position
of the more stable conformation of the substrate with
pseudoequatorial hydroxyl group, and if water is lost
stereoselectively from the pseudoaxial position before a
complete conformational equilibration of the formed gem-
diol has occurred, an excess of [18O] over [16O] ketone will
be formed.
of the label with the atmospheric oxygen had occurred,
consistent with a 16O atmospheric oxygen incorporation
followed by 18O or 16O re-expulsion. The absence of a
significant oxygen exchange between the ketone and H2O
was checked by control experiments.
Discu ssion
The present results show that the microsomal oxida-
tion of 1, chosen as a simple model substrate for allylic
alcohols, is catalyzed by the cytochrome P450 system and
leads exclusively to 2. Radical reactants do not appear
to contribute to this reaction, in analogy with the
oxidation of cyclohexanol to the corresponding ketone (3).
Several P450 isoforms, and particularly P450 2C11 and
2B1, are able to catalyze this reaction, although with
different rates and affinities.
An alternative explanation consists in a competition
between the gem-diol pathway a and the double hydrogen
abstraction pathways b and/or c. If the latter alternative
is correct, the mass spectrometric data allow to quantify
the ratio between these pathways to ca. 75:25. While it
is not possible, on the sole basis of the present data, to
distinguish between these two alternatives, it can be
observed that the cytochrome P450-catalyzed andros-
tenedione formation has been reported (18) to proceed
exclusively through the gem-diol pathway from epitest-
osterone, but through a combination of gem-diol and dual
hydrogen abstraction pathways from testosterone.
Several mechanistic pathways, which can be in part
distinguished by the use of [18O] labeled substrate, can
be evisaged for the oxidation of 1 (Scheme 1). In all cases
the reaction occurs through the abstraction of an hydro-
gen radical from the carbinol carbon by the perferryl
oxygen which is believed to be the ultimate oxidating
species of P450. The kinetic isotope effect, kH/kD ) 5.5,
measured for the oxidation of 1 and its [2H]-1 isotopomer
is in agreement with a primary isotope effect. However,
the value found in this work is lower than those reported
for primary intrinsic isotope effects related to other P450-
catalyzed hydroxylations (16) and then seems indicate
that the C(1) carbon-hydrogen bond breaking is only a
partially rate limiting step in the formation of 2. Fur-
thermore, considering that the substrate cannot escape
from the enzyme once the enzyme has been activated (16)
and that deuteration at the reaction site generally
produces a metabolic switching to some other product,
oxidation at another position, or water formation, the
absence of each of the three potential metabolites, cis-
2,3-epoxycyclohexanol, trans-2,3-epoxycyclohexanol, and
1-cyclohex-2-en-1-ol, suggests that water is the most
probable alternate product when [2H]-1 is used as sub-
strate.
The present data also show that the P450-catalyzed
oxidation of 1 is completely chemoselective, with complete
exclusion of double bond epoxidation. The presence of
the allylic electron withdrawing hydroxyl group seems
to favor, with respect to the double bond epoxidation, the
hydrogen atom abstraction mechanism leading to the
radical at C(1) whose subsequent fate has been discussed
above.
The investigation of the cytochrome P450-catalyzed
oxidation of other olefinic substrates bearing electrone-
gative substituents at the allylic positions is being
planned in order to check this interpretation. These
studies are expected to give a contribution to the ratio-
nalization of the factors affecting the formation of po-
tentially toxic metabolites such as epoxides during the
biotransformation of olefinic xenobiotics.
The formed radical should be very short-lived, because
no rearrangement to products other than 2 appears to
occur before it is rebound to the perferric hydroxide
radical (effectively a hydroxyl radical) to form a gem-diol,
which finally gives the ketone by water loss (path a). A
complete isotopic scrambling would be expected for this
Ack n ow led gm en t. This work was supported in part
by grants from Consiglio Nazionale delle Ricerche (CNR,
Roma) and Ministero dell’Universita` e della Ricerca

874 Chem. Res. Toxicol., Vol. 9, No. 5, 1996
Bellucci et al.
(9) Lowry, O. H., Rosebrough, N. J ., Farr, A. L., and Randall, R. J .
(1951) Protein measurements with the Folin phenol reagent. J .
Biol. Chem. 193, 265-275.
Scientifica e Tecnologica (MURST, Roma). We thank Dr.
A. Raffaelli for the GC-MS spectra.
(10) Omura, T., and Sato R. (1964) The carbon monoxide-binding
pigment of liver microsomes. J . Biol. Chem. 239, 2370-2378.
(11) Guengerich, F. P., Ghazi, A. D., Wright, T., Martin, M. V., and
Kamisky, L. S. (1982) Purification and characterization of liver
microsomal cytochrome P-450: Electrophoretic, spectral, catalytic,
and immunochemical properties and inducibility of eight isozymes
isolated from rats treated with phenobarbital or R-napthoflavone.
Biochemistry 21, 6019-6130.
(12) Fujita, S., Morimoto, R., Chiba, H., Kitani, K., and Suzuki, T.
(1989) Evaluation of the involvement of a male specific cytochrome
P-450 isozyme in senescence-associated decline of hepatic drug
metabolism in male rats. Biochem. Pharmacol. 38, 3225-3231.
(13) Ryan, D., and Levin, W. (1990) Purification and characterization
of hepatic microsomal cytochrome P-450. Pharmacol. Ther. 45,
153-239.
(14) A note of caution should be put forward about the estimation of
the kinetic data as they were obtained with substrate concentra-
tions not higher than 50 mM.
(15) Ruff, F., and Csimadia, I. G. (1994) in Organic Reactions,
Equilibria, Kinetics and Mechanism, pp 222-227, Elsevier, New
York.
(16) Harada, N., Miwa, G. T., Walsh, J . S., and Lu, A. Y. H. (1984)
Kinetic isotope effects on cytochrome P-450 catalyzed oxidation
reactions. Evidence for the irreversible formation of an activated
oxygen intermediate of cytochrome P-448. J . Biol. Chem. 259,
3005-3010.
Refer en ces
(1) Nelson, D. R., Kamataki, T., Waxman, D. J ., Guengerich, F. P.,
Estabrock, R. W., Feyeriesen, R., Gonzalez, F. J . Coon, M. J .
Gunsalus, I. C., Gotoh, O., Okuda, K., and Nebert, D. W. (1993)
DNA Cell Biol., 12, 1-51.
(2) Ortiz de Montellano (1985) in Bioactivation of Foreign Compounds
(Ortiz de Montellano, Ed.) pp 121-155, Academic Press, New
York.
(3) Gorsky, L. D., and Coon, M. J . (1985) Evaluation of the role of
free hydroxyl radicals in the cytochrome P-450-catalyzed oxidation
of benzene and cyclohexanol. Drug. Metab. Dispos. 13, 169-174.
(4) Guengerich, F. P., and Shimada, T. (1991) Oxidation of toxic and
carcinogenic chemicals by human cytochrome P-450 enzymes.
Chem. Res. Toxicol. 4, 391-407.
(5) Lippi, A., Gervasi, P. G., Bellucci, G., Marioni, F., Luzzani, F.,
and Ventura, P. (1989) Effects of trans-sobrerol on drug metabo-
lizing enzymes in the rat. Xenobiotica 19, 823-832.
(6) Bellucci, G., Chiappe, C., Lippi, A., Marioni, F., and Gervasi, P.
G. (1991) The cytochrome P-450 catalyzed oxidation of 1-meth-
ylcyclohexene. Competition between hydroxylation and epoxida-
tion and absolute stereochemistry of epoxidation. Biorg.-Med.
Chem. Lett. 1, 121-124. Bellucci, G., Chiappe, C., Lippi, A.,
Marioni, F., Ventura, P., and Gervasi, P. G. (1992) 1-Methylcy-
clohexene metabolism by microsomes and purified cytochrome
P-450 IIB1 from rat liver. Biochemistry (Life Sci. Adv.) 11, 107-
116.
(7) Gervasi, P. G., Citti, L., Turchi, G., Bellucci, G., Berti, G.,
Mastrorilli, E., and Tortello, M. P. (1982) The metabolism of 1,3-
cyclohexadiene by liver microsomal mono-oxygenase. Xenobiotica
12, 517-526.
(8) Menicagli, S., Longo, V., Mazzaccaro, A., and Gervasi, P. G. (1994)
Microsomal metabolism of N,N-diethylacetamide and N,N-di-
methylacetamide and their effects on drug-metabolizing enzymes
of rat liver. Biochem. Pharmacol. 48, 717-726.
(17) Guengerich, F. P., and Kim, D. H. (1991) Enzymatic oxidation of
ethyl carbamate to vinyl carbamate and its role as an intermedi-
6-ethenoadenosine. Chem. Res. Toxicol.
ate in the formation of 1,N
4, 413-421.
(18) Wood, A. W., Swinney, D. C., Thomas, P. E., Ryan, D. E., Hall,
P. F., Levin, W., and Garland, W. A. (1988) Mechanism of
androstenedione formation from testosterone and epitestosterone
catalyzed by purified cytochrome P-450b. J . Biol. Chem. 33,
17322-17332.
TX9600053