In the biosynthesis of steroid hormones, P450c17
is the single enzyme that catalyzes both the 17
-hydroxylation of
21-carbon steroids and the 17,20-lyase activity that cleaves the
C17-C20 bond to produce C19
sex steroids. Cytochrome b5 augments the
17,20-lyase activity of cytochrome P450c17 in vitro, but
this has not been demonstrated in membranes, and the mechanism of this
action is unknown. We expressed human P450c17, human
P450-oxidoreductase (OR), and/or human cytochrome
b5 in Saccharomyces cerevisiae and analyzed the 17
-hydroxylase and 17,20-lyase activities of the resulting yeast microsomes. Yeast expressing only P450c17 have 17
-hydroxylase and trace 17,20-lyase activities toward both
4 and
5 steroids. Coexpression of human
OR with P450c17 increases the Vmax of both the
17
-hydroxylase and 17,20-lyase reactions 5-fold; coexpression of
human b5 with P450c17 also increases the
Vmax of the 17,20-lyase reactions but not of
the 17
-hydroxylase reactions. Simultaneous expression of human
b5 with P450c17 and OR, or addition of purified
human b5 to microsomes from yeast coexpressing
human P450c17 and OR, further increases the
Vmax of the 17,20-lyase reaction without
altering 17
-hydroxylase activity. Genetically engineered yeast and
mixing experiments demonstrate that OR is both necessary and sufficient
for microsomal 17,20-lyase activity. Addition of purified human
holo-b5, apo-b5, or
cytochrome c to microsomes containing both human P450c17
and OR demonstrate that the stimulatory action of
b5 does not require electron transfer from
b5 to P450c17. These data suggest that human
b5 acts principally as an allosteric effector
that interacts primarily with the P450c17·OR complex to stimulate
17,20-lyase activity.
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INTRODUCTION |
Among the many chemical transformations catalyzed by cytochrome
P450 enzymes, steroid hormone hydroxylations, and cleavages are of
particular interest because of their mechanistic complexities and
essential roles in physiology (1). P450c17 catalyzes both 17
-hydroxylase and 17,20-lyase activities (2) (for review see Ref.
3) and also has a modest degree of 16
-hydroxylase activity (4). In
human beings, the 17
-hydroxylase reaction leads to the
glucocorticoid, cortisol, and the subsequent 17,20-lyase reaction leads
to precursors of sex steroids. As the sole pathway leading to
biosynthesis of circulating sex steroids, the regulation of this
17,20-lyase activity is central to understanding the developmental regulation of dehydroepiandrosterone sulfate
(DHEA)1 with adrenarche and
aging, and to the pathogenesis of the polycystic ovary syndrome (3).
The 17,20-lyase activity, involving the oxidative cleavage of a
carbon-carbon bond, is regulated in a tissue-specific and
developmentally programmed manner by factors such as the abundance of
the electron donor flavoprotein P450-oxidoreductase (OR) (5, 6), the
co-existence of 3
-hydroxysteroid dehydrogenase and P450c21 (7), and
post-translational modification of P450c17 (8).
To perform catalysis, P450c17, like all other microsomal P450
oxygenases, must receive two electrons from NADPH via OR. Cytochrome b5 has also been implicated as a component of
the 17,20-lyase reaction, as b5 augments
17,20-lyase activity and occasionally 17
-hydroxylase activity of
P450c17 in reconstituted systems (9, 10); however, our laboratory could
not confirm this effect in transfected monkey kidney COS-1 cells (5).
Inconsistencies in the animal species of P450c17, OR, and
b5 used in previous studies preclude
extrapolation of the available biochemical data to human adrenal and
gonadal physiology; furthermore, the mechanism(s) of these reported
b5-mediated increases in 17,20-lyase activity remain unknown.
Among the various systems developed to study mammalian cytochromes
P450, transfection of genetically modified yeast cells provides the
opportunity to study the activities of a cytochrome P450 in the
presence of various combinations of electron transfer proteins in the
native microsomal environment (11). To clarify the function of
cytochrome b5 in 17,20-lyase activity, we
systematically varied the abundance of putative electron transfer
proteins in yeast microsomes containing human P450c17. We find that
human, but not yeast cytochrome b5 can
selectively augment the rate of the 17,20-lyase reaction by more than
10-fold. However, this augmentation requires OR and occurs without
electron transfer to or from cytochrome b5.
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EXPERIMENTAL PROCEDURES |
Yeast Strains and Expression Vectors--
Wild type yeast
strains W303A (Y150WT) (leu2-3, 112; his3-11, 15;
trp1-1; ade2-1; ura3-1; mat a) and
W303B (JC104) (trp1-1; ura3-1; ade2-1;
can1-100; mat
) were generous gifts of Drs. Gregory Petsko and Ira Herskowitz. Engineered yeast strains, W(B), W(hR), and
W(B
), generated by targeted disruption of the yeast CPR1 or YCY b5 loci (11-14) and the yeast expression
vectors V10 and V60 (11) were generous gifts of Dr. Denis Pompon (CNRS,
Gif-sur-Yvette, France). Human P450c17 cDNA (15) was PCR amplified
with Pfu polymerase (Stratagene, La Jolla, CA) using primers
c17-S-1 and c17-AS-1 (Table I) and pECE-c17 (16) as template. The
resulting PCR product was digested with BamHI and
EcoRI, facilitating directional cloning into complementary
ends of BglII-EcoRI digested V10 vector, destroying the BglII site and placing the P450c17 cDNA
under the control of the constitutive pgk promoter,
producing vector V10-c17. V60 was modified by disruption of the
ura3 gene by digestion with NcoI, blunting of the
staggered ends, and religation, yielding vector pYeSF1. Using the
unique BamHI site at the 5
end of the pgk
promoter in V10, a BamHI-EcoRI fragment of the
V10-c17 plasmid was cloned into BamHI-EcoRI
digested pYeSF1 to provide ade2 complementation to the
pgk-regulated P450c17 (vector pYeSF2-c17). Vector cDE2, used
to generate doubly transformed yeast with either V10-c17 or pYeSF2-c17,
was a generous gift from Dr. Ira Herskowitz (17). Human
P450-oxidoreductase cDNA was PCR amplified from pECE-OR (5) using
primers OR-S-1 and OR-AS-1 (Table I). The
5
-PCR primer contained two silent base pair changes from the wild type sequence to remove hairpin structures surrounding the translation initiation codon (12) which can inhibit transcription and translation in yeast (18). Cytochrome b5 cDNA was
generated by reverse transcription of total human testicular RNA using
random hexamers followed by PCR amplification with primers
b5-S-1 and b5-AS-1 (Table
I) based on the human b5 cDNA sequence (19).
The human b5 and OR cDNAs were then cloned
into the EcoRI site of cDE2 vector under control of the
constitutive adc1 promoter with a trp1 selectable
marker. The accuracy and orientation of all constructions were
confirmed by DNA sequencing.
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Table I
PCR primers
Restriction sites (BamHI or EcoRI) are
underlined, and ATG start codons are in bold type. Silent base pair
changes to eliminate hairpin loop formation in OR-S-1 are underlined
and in bold.
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Yeast Transformation and Growth--
Yeast were transformed
using 700 µl of 40% polyethylene glycol 3350, 0.1 M
lithium acetate, 10 mM Tris-HCl (pH 8), 1 mM
EDTA to transform 106 yeast in 100 µl of 0.1 M lithium acetate, 10 mM Tris-HCl (pH 8), 1 mM EDTA with 1-2 µg of plasmid DNA and 50 µg of
denatured herring sperm carrier DNA (20). Cells were washed in 100 µl of 1 M sorbitol before final resuspension in 100 µl of 10 mM Tris-HCl (pH 8), 1 mM EDTA and plating onto
selective media. All transformations introduced two plasmids
simultaneously: the first, expressing P450c17, was either V10-c17 or
pYeSF2-c17; the second was cDE2, containing no cDNA insert or the
cDNA for either human OR or b5. Thus,
P450c17 expression was always under the control of the constitutive pgk promotor, and all yeast producing different combinations
of electron donor proteins were grown in the same culture medium. For
microsome preparations, transformed yeast were cultured in minimal SD
media containing 20 g/liter D-glucose or
D-galactose, 1.7 g/liter yeast nitrogen base without amino
acids or ammonium sulfate (Difco, Detroit, MI), 5 g/liter ammonium
sulfate, and supplemented with the requisite combination of 10 mg/liter
leucine, 15 mg/liter adenine, and 10 mg/liter histidine (11).
Microsome Preparation and Characterization--
Yeast cells
harvested at a density of 4.5-6 × 107 cells/ml were
disrupted by manual breakage with glass beads (450-600 micron) for 5 min (11). The breakage was stopped at 1-min intervals, and cells were
iced for 30 s; 3 µl of 0.5 M ethanolic
phenylmethylsulfonyl fluoride was added after the first minute of
breakage. For a typical 300-ml culture, crude extracts and beads were
washed twice with 5-7 ml of 50 mM Tris-HCl (pH 8), 1 mM EDTA, 0.4 M sorbitol, and the cellular
debris was collected by centrifugation at 4 °C twice for 10 min at
14,000 × g. Microsomes were pelleted by centrifugation at 4 °C for 45 min at 100,000 × g and were
resuspended in 50 mM Tris-HCl (pH 8), 1 mM
EDTA, 20% glycerol at 5-20 µg/µl total protein. Preparations were
homogenized by shearing microsomes through a 27-gauge needle 10 times
and were kept frozen at
70 °C until needed. Human adrenal
microsomes were prepared from excess surgical tissue as described
(8).
Microsomal proteins were quantitated colorimetrically. Immunoblotting
on polyvinylidene difluoride membranes (Millipore, Bedford, MA) was
performed with rabbit antiserum to human P450c17 (5) or to human OR
(generously provided by Prof. C. Roland Wolf, Imperial Cancer
Institute, Dundee, United Kingdom) using secondary antibody-peroxidase conjugate and ECL reagents (Amersham, Arlington Heights, IL) and with
goat antiserum to human b5 (Oxford Biomedical,
Rochester Hills, MI) using secondary antibody-peroxidase conjugate
(Santa Cruz Biotechnology, Santa Cruz, CA) and ECL reagents. Microsomal P450 and cytochrome b5 contents were measured
spectroscopically (21) using either a Cary 3E or a Shimazdu UV160U
spectrophotometer. P450 oxidoreductase activity was measured as
described (22).
P450c17 Enzyme Assay--
Microsomes were assayed under initial
rate kinetics by preincubation in 50 mM potassium phosphate
buffer (pH 7.4) with 0.5-5 µM steroid (added in 4 µl
of ethanol) in 200 µl total volume at 37 °C for 2 min before the
addition of 1 mM NADPH to start the reaction. Each reaction
contained either 20,000 cpm of [3H]pregnenolone,
[3H]17
-hydroxypregnenolone (NEN Life Science Products
Inc., Boston, MA), or [3H]17
-hydroxyprogesterone
(Amersham) or 10,000 cpm of [14C]progesterone (NEN Life
Science Products). Steroids were extracted with 400 µl of ethyl
acetate/isooctane (1:1), concentrated under nitrogen, separated by thin
layer chromatography (Whatman PE SIL G/UV silica gel plates, Maidstone,
Kent, UK) using 3:1 chloroform/ethyl acetate, and quantitated as
described (23). Purified recombinant human cytochrome
b5 (Pan Vera, Madison, WI), apo-human cytochrome b5, or horse heart cytochrome c
(Sigma) were included in incubations as indicated. Apo-cytochrome
b5 was prepared from the Pan Vera holo-cytochrome b5 as described (24), and absent
electron transfer properties of the resulting material was confirmed by
difference spectroscopy. Kinetic behavior was approximated as a
Michaelis-Menten system for data analysis, and all error bars shown
represent standard deviations.
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RESULTS |
Yeast Transfection and Microsome Characterization--
The
capacity of b5 to increase the 17,20-lyase
activity of P450c17 has been shown by several laboratories using
purified, reconstituted protein systems (9, 25), but this phenomenon has not been observed in intact cell and microsome preparations (5).
The development of "humanized" yeast strains (12) that express both
P450c17 and selected electron donor proteins has enabled us to dissect
this problem without detergent solubilization of individual
components.
To study the effects of human OR and b5 on human
P450c17 activities in yeast microsomes, parental yeast strain W303B was
doubly transfected with vector V10 expressing human P450c17 and with vector cDE2 expressing either the cDNA for human OR or
b5 (or empty vector). Microsomes from these
transfectants were characterized and used for kinetic studies;
microsomes were also prepared from transfections using the same vectors
with the cDNA inserts exchanged. Both sets of transfections were
also performed using yeast strain W303A; abbreviated kinetic
experiments using these W303A-derived microsome preparations yielded
qualitatively similar results as did microsomes and spheroplasts
prepared from the W303B transfectants; thus, W303B doubly transfected
with V10-c17 plus cDE2 (for expressing an electron donor) was used in
all subsequent experiments.
P450 content, total OR (cytochrome c reductase activity),
and total cytochrome b5 content were similar
among the three microsome preparations from co-transfected W303B yeast
(Table II). Essentially all of the P450
is from P450c17, whereas cytochrome c reductase activity and
total cytochrome b5 content were similar in all
transfectants, indicating that endogenous yeast OR and
b5 are the predominant electron transfer
proteins in these microsomes. Comparable expression of P450c17 was
demonstrated in all samples by Western blotting, and human OR and
b5 were detected only in samples from yeast
containing their respective cDNAs, as expected (Fig.
1).

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Fig. 1.
Immunoblot of yeast microsomal proteins.
Denatured microsomal proteins (2-5 µg) from yeast transfected with
the various vectors as labeled at the top of the figure were
electrophoresed through a 10% SDS-polyacrylamide gel, blotted to
polyvinylidene difluoride, and probed with antisera to P450c17 and OR.
Expression of human P450c17 (c17) is comparable in all
samples, but human OR is expressed only in yeast containing the OR
expression vector. Immunoblotting with antisera to rat or rabbit
b5 showed aggregation and cross-reactivity (not
shown). Dash ( ) indicates use of empty vector.
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Kinetics--
To determine how the presence of human OR and/or
b5 alters the activities of human P450c17 in
yeast microsomes, we measured apparent Km and
Vmax values for both 17
-hydroxylase and
17,20-lyase reactions for
5 and
4
substrates (Table III). Lineweaver-Burk
plots (Fig. 2) show that yeast
transfected with human P450c17 alone perform both 17
-hydroxylase and
17,20-lyase reactions despite the absence of human electron transfer
proteins, indicating that the endogenous yeast OR can couple with human
P450c17, as has been shown for bovine P450c17 (26, 27). In this system,
however, 17,20-lyase activity is very low but not absent, as reported
for bovine P450c17 (26). Co-expression of human OR substantially
increases both activities. Using the
5 steroid
substrates pregnenolone and 17
-hydroxypregnenolone, co-expression of
human OR raises the Vmax for both the
17
-hydroxylase and 17,20-lyase reactions 5-fold. In yeast
microsomes, the apparent Km for both pregnenolone
and 17
-hydroxypregnenolone is about 1 µM, and
co-expression of human OR lowers the Km to below 0.5 µM, suggesting that the association of human P450c17 and
OR may increase the affinity of P450c17 for
5
substrates. The presence of OR also alters the orientation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in the substrate-binding pocket of P450 2D6 (28), suggesting that OR may participate in
substrate discrimination by P450 enzymes. DHEA formed by microsomes containing both human P450c17 and OR is metabolized further to a more
polar compound, possibly 16
-hydroxy-DHEA, the major DHEA metabolite
of a bovine P450c17/rat OR fusion protein (29). Human OR markedly
stimulated both activities without a significant change in total
cytochrome c reductase activity, indicating that yeast OR is
an inefficient electron donor for P450c17 and does not significantly interfere with catalysis in the presence of human OR.
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Table III
Kinetic constants
Apparent Km and Vmax values were
obtained from linear regression analysis of the data in Fig. 2.
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Fig. 2.
Lineweaver-Burk plots of 17 -hydroxylase
and 17,20-lyase activities. Lines were derived from
least-squares fit to data points (r2 > 0.92 for
all lines). The apparent Km and
Vmax values obtained from these data are shown
in Table III. Microsomes were prepared from W303B yeast
co-transfected with V10-c17 and the empty cDE2 vector
(squares), V10-c17 and cDE2-OR (circles), or V10-c17 and cDE2-b5 (triangles).
Panel A, incubations with 5 pregnenolone.
Panel B, incubations with 5
17 -hydroxypregnenolone. Panel C, incubations with
4 progesterone. Panel D, incubations with
4 17 - hydroxyprogesterone.
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When
4 substrates are used, human P450c17 efficiently
catalyzed the conversion of progesterone to 17
-hydroxyprogesterone, but the conversion of 17
-hydroxyprogesterone to androstenedione was
much less efficient than the corresponding conversion of
17
-hydroxypregnenolone to DHEA (Table III). The slow turnover of
17
-hydroxyprogesterone by human P450c17 explains why circulating
androgens in humans derive principally from the isomerization and
reduction of DHEA rather than by cleavage of 17
-hydroxyprogesterone
to androstenedione, the predominant pathway in rodents. Guinea pig
P450c17, for example, preferentially converts progesterone to
androstenedione, some of which is sequentially metabolized without
dissociation of the intermediate 17
-hydroxyprogesterone from
the active site (30, 31). Co-expression of human OR similarly increases
the Vmax of both activities toward
4 steroids but without a significant change in apparent
Km values (Table III). A second, more polar product,
presumably 16
-hydroxyprogesterone (4, 5, 32), constitutes
~20-25% of the products when
4-progesterone is the
substrate with all microsomes tested.
Co-expression of human b5 with human P450c17
increases Vmax 10-fold for the 17,20-lyase
reaction but not for the 17
-hydroxylase reaction with both
5 and
4 substrates and does not change
the apparent Km for any substrate tested (Table
III). Although human OR improves the catalytic efficiency of P450c17 in
yeast microsomes, both by lowering the Km of
5 substrates and increasing the
Vmax for all reactions, the sole effect of human
b5 is to augment the Vmax
for the 17,20-lyase reactions. Our results generally agree with those
obtained with reconstituted recombinant human P450c17 and rat OR,
except that rat b5 approximately doubles the
rate of hydroxylation of
5-pregnenolone but not of
4-progesterone (25). Differences in species of origin of
the OR and b5 used may explain some differences
in the results obtained in the two systems, as well as subtle
differences in the activities of microsomal and detergent-solubilized
proteins.
Activities in the Absence of Yeast OR or Yeast Cytochrome
b5--
The experiments described above were performed in
the presence of endogenous yeast OR and b5 in
the microsome preparations. To determine whether the yeast electron
donors influence human P450c17 activities, we expressed human P450c17,
with human OR or b5, in engineered yeast strains
lacking the endogenous yeast OR or b5 genes.
When human P450c17 and OR were coexpressed in yeast strain W(B
),
which lacks the yeast homolog of the human b5
gene (13), the resulting microsomes contained 85% of the 17
-hydroxylase activity and 73% of the 17,20-lyase activity of microsomes from W303B yeast (Fig.
3A). The 17,20-lyase activity was minimally affected by the absence of yeast
b5, demonstrating that OR is both necessary and
sufficient to confer both 17
-hydroxylase and 17,20-lyase activity to
human P450c17 in yeast microsomes.

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Fig. 3.
Human P450c17 activities in engineered yeast
strains. Panel A, 17 -hydroxylase (open bars)
and 17,20-lyase (hatched bars) activities in microsomes
prepared from yeast strains W(B ), lacking yeast
b5, or from W303B yeast using 1 µM
steroid. Note the 10-fold difference in the scales of hydroxylase
activity (left) and lyase activity (right). Error
bars (±S.D.) are too tight to be seen in three of the four bars.
Panel B, P450c17 activities in microsomes from W(B) yeast
(lacking yeast OR) co-transfected with V10-c17 and empty cDE2 vector or
cDE2-OR. Clones were grown in glucose, producing trace (lo)
amounts of human b5, or in galactose, inducing
high (hi) amounts of b5. Incubations
contained 1 µM steroid and 25 or 125 µg of microsomal
protein to assay 17 -hydroxylase activity (1 h incubation,
lanes 1-4) or 17,20-lyase activity (4 h incubation,
lanes 5-8), respectively. Panel C,
Lineweaver-Burk plot of 17,20-lyase activity in microsomes from W(B)
yeast co-transfected with pYeSF2-c17 and cDE2-OR. Microsomes were
prepared from the same yeast clone expressing trace human
b5 (grown in glucose, squares), or
expressing high b5 (grown in galactose,
circles). Apparent Km and
Vmax values were derived from least-squares fits
to the data.
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To confirm that OR was required for catalysis, we expressed human
P450c17 in strain W(B), in which the endogenous yeast OR locus is
replaced by the human b5 cDNA under the
control of the inducible Gal10/Cyc1 promoter (12). No 17
-hydroxylase
or 17,20-lyase activity is present in microsomes prepared from W(B)
yeast transfected with human P450c17 and empty cDE2 vector, but both
activities are restored by co-transfection of human OR (Fig.
3B). When W(B) yeast, transfected with both human P450c17
and OR, were grown in galactose to induce expression of human
b5 as well, the presence of human
b5 increased the Vmax of
the 17,20-lyase reaction using 17
-hydroxypregnenolone from 0.14 min
1 to 1.1 min
1 but did not change the
apparent Km (0.3 µM) (Fig. 3, B and C). This induction of human
b5 did not significantly change 17
-hydroxylase activity, reflected by comparable pregnenolone consumption (Fig. 3B, lanes 3 and 4), but the
17
-hydroxypregnenolone formed in the presence of high amounts of
human b5 was rapidly converted to DHEA, so that
little 17
-hydroxypregnenolone accumulated (Fig. 3B, lane
4).
Effect of Exogenous Soluble b5 on 17
-Hydroxylase and
17,20-Lyase--
Exogenously added soluble b5
can influence other P450 reactions in yeast microsomes (11); therefore,
we added purified human b5 to yeast microsomes
containing human P450c17. Although 17
-hydroxylase activity against
5-pregnenolone or
4-progesterone was not
changed (Fig. 4, A and
C), 17,20-lyase activity against 17
-hydroxypregnenolone
was increased up to 10-fold in microsomes that did not already contain
human b5 (Fig. 4B). Purified b5 also increased 17,20-lyase activity toward
17
-hydroxyprogesterone, but only about 2-fold (Fig. 4D).
These data demonstrate that yeast b5 can neither
support nor stimulate human P450c17 activities, as found for other
human P450s (12). Furthermore, our results show that the only effect of
human b5, either added in solution or
coexpressed into microsomes, is to increase the rate of the 17,20-lyase
reactions, and that this action of b5 requires
the presence of yeast or human OR.

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Fig. 4.
Effect of exogenously added human
b5 on P450c17 activities. Microsomes were
prepared from yeast strain W303B expressing human P450c17 and
co-transfected with cDE2 vector expressing no protein, OR, or
b5 as indicated. The indicated steroid for each panel (5 µM) was incubated with (+) and without ( ) 1 molar equivalent of exogenously added purified human
b5 per molar equivalent of P450c17. Incubations
contained 5 pregnenolone (panel A),
5 17 -hydroxypregnenolone (panel B),
4 progesterone (panel C), and
4 17 -hydroxyprogesterone (panel D). Note
the different scales in each panel.
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The results described above do not exclude a contribution of human
b5 as the donor of the second of the two
electrons in the P450 catalytic cycle, as has been suggested (33, 34).
If b5 functions as the donor of the second
electron, b5 should support catalysis by
transporting electrons either from a reducing agent (sodium dithionite)
or from NADPH-reduced OR to microsomes containing P450c17 that has
already been reduced with the first electron. Dithionite, which can
provide one electron to either P450c17 or b5,
does not support catalysis in microsomes containing both human P450c17
and b5, but dithionite does not abolish
catalysis when the second electron is provided to P450c17 from NADPH
via OR (Fig. 5A). To confirm
this observation, we attempted to reconstitute 17,20-lyase activity by
transferring electrons from NADPH to one pool of microsomes containing
human OR (and no P450c17), then to soluble human
b5 as an electron conduit, and finally to human P450c17 in another pool of microsomes lacking OR. Soluble
b5 was first reduced with NADPH by microsomes
containing OR (35), and then added to microsomes lacking yeast OR
(strain W(B)) but containing human P450c17 alone (lane 1),
human P450c17 and OR (lane 2), or human P450c17 and
b5 (but no OR, lane 3), all of which
had been preincubated with 17
-hydroxypregnenolone and dithionite to
provide the first electron to P450c17. Microsomes lacking human OR
converted only a trace of 17
-hydroxypregnenolone to DHEA under these
conditions, but microsomes containing both human P450c17 and OR could
use the added NADPH to convert substrate to DHEA (Fig. 5B).
These results confirm that b5, reduced either by
dithionite or OR, cannot provide sufficient electron transfer to
P450c17 to support significant 17,20-lyase activity. Therefore, these
data suggest that the mechanism by which b5
enhances 17,20-lyase activity does not involve electron transfer.

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Fig. 5.
Reconstitution of human P450c17 activities
using sodium dithionite and cytochrome
b5. Panel A, 17 -hydroxylase and 17,20-lyase activities in microsomes from W(B) yeast (lacking yeast OR)
co-transfected with pYeSF2-c17 and cDE2-b5 (0.1 mg of protein containing 9 pmol of P450 and 42 pmol of
b5). Microsomes were incubated with either 1 µM pregnenolone (lanes 1 and 3) or 1 µM 17 -hydroxypregnenolone (lanes 2 and 4) and a saturating portion of solid sodium dithionite
(21). Incubations for lanes 3 and 4 also
contained NADPH and additional microsomes (16 µg of protein)
containing human P450c17 (1 pmol) and OR. Panel B, 17,20-lyase activity in microsomes containing 5 pmol of human P450c17
alone (lane 1), with human OR (lane 2), or with
human b5 (lane 3). Microsomes were
preincubated with 1 µM 17 -hydroxypregnenolone and
solid sodium dithionite in 100 µl, and the reactions were started by
adding an equal volume of a second incubation containing soluble human
b5 (50 pmol) plus microsomes containing human OR but no P450c17 (50 µg of protein, cytochrome c reductase
activity, 104 ± 15 nmol/min/mg protein) and NADPH (2 mM) to reduce the soluble b5.
Migrations of 5 steroids are indicated (S,
17 -hydroxypregnenolone standard).
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How Does Human Cytochrome b5 Augment 17,20-Lyase
Activity?--
To explore the mechanism by which
b5 increases 17,20-lyase activity, we assayed
the 17,20-lyase activity of microsomes containing constant, high
amounts of P450c17 and OR and varying amounts of b5. A sharp increase in 17,20-lyase activity was
observed when the molar ratio of b5 to P450c17
approached 1:1 (Fig. 6A).
Activity reached a maximum at ratios of b5 to
P450c17 between 10:1 and 30:1; however, further addition of human
b5 progressively inhibited 17,20-lyase activity
in both yeast and human adrenal microsomes. If human
b5 was acting as the preferred electron donor,
17,20-lyase activity should saturate and remain constant rather than
fall at high b5/P450c17 ratios. Similarly, when
we examined the influence of b5 on the
17,20-lyase activity of microsomes containing very small amounts of
human OR and no yeast OR (strain W(hR) transfected with P450c17 grown
to high density in glucose), maximal stimulation occurred at a
b5/P450c17 ratio between 1:1 and 3:1, and higher ratios were again inhibitory (Fig. 6A). Thus, the influence
of human b5 changes dramatically as the
abundance of human OR and the b5/P450c17 ratio
are varied. These data suggest that b5 does not
function as an electron donor, but instead exerts some other action,
perhaps facilitating electron transfer from OR to P450c17 or improving
coupling efficiency, as has been suggested for other P450 reactions
stimulated by b5 (24, 36).

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Fig. 6.
Activation and inhibition of 17,20-lyase
activity by cytochromes b5 and
c. Conversion of 0.5 µM
17 -hydroxypregnenolone to DHEA by human adrenal microsomes
(triangles), yeast microsomes with high amounts of human OR
(squares), or yeast microsomes with low amounts of human OR
(circles) plus the indicated molar ratios of human
holo-b5 (panel A), human
apo-b5 (panel B), or horse heart cytochrome c (panel C). Activity is expressed as
the percent of conversion by the microsomes alone. The yeast microsomes
were prepared from strain W(hR) either co-transfected with pYeSF2-c17 plus cDE2-OR and grown in galactose, yielding the microsomes with high
amounts of human OR, or co-transfected with pYeSF2-c17 plus empty cDE2
vector and grown in glucose, yielding the microsomes with low amounts
of human OR (cytochrome c reductase activities of 223 and 12 nmol/min/mg protein, respectively).
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Inhibition of enzymatic activity at high
b5/P450c17 ratios, a phenomenon also observed in
guinea pig adrenal microsomes (10), could result from a second,
inhibitory b5-binding site on P450c17 or from a
competition between b5 and P450c17 for electrons
from limiting amounts of OR. Cytochrome c, which is also a
substrate for reduction by OR (37), also inhibits 17,20-lyase activity at molar ratios above 10:1, the same molar ratios at which
b5 becomes inhibitory (Fig. 6C).
Inhibition by equivalent molar ratios of cytochrome c to
P450c17 is consistent with b5 competing with P450c17 for reduction when OR is limiting, but "reverse" electron transfer from P450c17 to b5 (38) may also
contribute to the inhibition observed at higher
b5/P450c17 ratios. These data suggest that
electron transfer from OR to b5 is actually detrimental to 17,20-lyase activity. Therefore, we determined whether human
apo-b5, which lacks the heme and hence cannot
participate in electron transfer, modulates 17,20-lyase activity
differently than human holo-b5. In microsomes
containing either low or high amounts of human OR, molar ratios of
apo-b5 to P450c17 between 1:1 and 10:1 augment
17,20-lyase activity (Fig. 6B). Unlike the data with
holo-b5, the stimulatory effect of
apo-b5 remains constant rather than falling at
higher b5/P450c17 ratios. These results exclude
direct electron transfer from b5 as the
principal means by which b5 augments 17,20-lyase
activity and suggest that b5 exerts a saturable,
allosteric effect on the P450c17·OR complex.
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DISCUSSION |
The 17,20-lyase/17
-hydroxylase ratio in the human adrenal rises
dramatically with the onset of adrenarche at age 8-10, reaches maximal
values at age 25-35, and then falls progressively with aging (39); as
these phenomena occur only in human beings and great apes (40), their
study is difficult. These selective, physiologic, developmentally
programmed changes in human adrenal 17,20-lyase activity imply
regulatory mechanisms beyond transcription of P450c17 or OR (3, 8).
Most P450 enzymes catalyze multiple reactions, but the ratio of their
activities remains fixed. The developmentally and possibly hormonally
programmed changes in the ratio of 17,20-lyase to 17
-hydroxylase
activities of human P450c17 provide a unique system for studying the
differential regulation of two reactions catalyzed by a single P450
enzyme.
An augmentation of the 17,20-lyase activity of P450c17 by
b5 has been observed in vitro (9, 25)
but was not seen in transfected COS-1 cells (5), possibly because the
endogenous b5 in those cells was sufficient to
stimulate 17,20-lyase activity maximally. Thus, it has not been clear
how or if b5 regulates human P450c17 activities
in vivo. The use of microsomes from yeast engineered to
express human P450c17, OR, or b5 from inducible
promoters permits the quantitative manipulation of each protein in a
membrane environment that should simulate events in vivo.
This permits greater experimental flexibility than the use of
bicistronic plasmids (41), fusion proteins (29), or viral vectors (42),
and obviates concerns about the relevance of data from
detergent-solubilized systems to in vivo systems.
Titration experiments with purified human
holo-b5, apo-b5, and
cytochrome c showed that the stimulatory effect of
b5 on 17,20-lyase activity is not mediated by
electron transfer from b5 and suggest that
b5 exerts an allosteric effect on the
P450c17·OR complex. This proposed mechanism could explain three
observations from other laboratories. First, b5
facilitates electron transfer from OR to P450 3A4 only when all three
proteins are premixed before adding NADPH and substrate, but not when
b5 is premixed with P450 3A4 and added to OR,
NADPH, and substrate in stop-flow experiments (24). These data
suggested that the stimulatory action of b5 on
testosterone 6
-hydroxylation by P450 3A4 was an allosteric effect
and was not mediated by an action of b5 as an
alternate electron donor (24). Second, b5 is a
more potent stimulator of 17,20-lyase activity when the abundance of OR
is low, and this stimulation is quite sensitive to small changes in
these low amounts of OR (10). Our results corroborate these studies and
suggest that b5 interacts primarily with the
P450c17·OR complex and not with P450c17 alone. Third, the
redox-active core 1 segment of porcine b5 alone
cannot augment the 17,20-lyase activity of human P450c17 (43),
consistent with our findings that electron transfer from human
b5 is not required to stimulate 17,20-lyase
activity.
Three conclusions about human physiology emerge from our analysis of
the kinetics of human P450c17. First, human androgen biosynthesis
proceeds predominantly through the pathway 17
-hydroxypregnenolone
DHEA
androstenedione, rather than through the pathway
17
-hydroxypregnenolone
17
-hydroxyprogesterone
androstenedione. The pathway via DHEA predominates because the apparent
Km for
4 17
-hydroxyprogesterone is
about 10-fold higher and its Vmax is one-tenth
as fast as the corresponding values for
5
17
-hydroxypregnenolone. Thus, the catalytic efficiency
Vmax/Km for the 17,20-lyase
reaction is nearly 100-fold greater for
5
17
-hydroxypregnenolone than for
4
17
-hydroxyprogesterone. Second, significant androgen biosynthesis via the
4 pathway can only occur in the presence of very
high
4 17
-hydroxyprogesterone concentrations, as
found in untreated patients with 21-hydroxylase deficiency (44). Third,
considerable microsomal 17,20-lyase activity is found even in the
complete absence of b5; therefore,
b5 deficiency cannot cause a syndrome of
complete 17,20-lyase deficiency (23) as has been suggested (45).
The structural nature of the interaction of P450c17 with OR is not
known, but the x-ray crystal structures of rat OR (46) and P450-BMP
(47) provide useful clues. The redox-partner binding site for P450-BMP,
a Type II (microsomal) P450, comprises the surface surrounding a
depression in the "proximal" face of the protein that extends down
to the face of the heme opposite the substrate-binding pocket (47, 48).
This crevasse is lined on one side with positively charged residues
from the J
and K helices (in P450-BMP, lysines 325, 328, and 331)
which appear to participate in electrostatic pairing with negatively
charged residues in OR. Molecular modeling shows that human P450c17 has a similarly located crevasse of positively charged residues that interact with redox partners (23). The electron-donating FMN moiety of
rat OR also lies at the base of a concave cleft formed by the
butterfly-shaped apposition of the FMN and FAD domains (46). However,
the FMN domain joins the remainder of the protein via a disordered,
flexible hinge that must flex about 90° for the FMN moiety to extend
out from the concave cleft of OR (46) to approach the concave
redox-partner binding site of P450c17 (23).
Because b5 normally participates in redox
reactions such as methemoglobin reduction (49) and stearyl-CoA
desaturation (50) and can serve as an alternate electron donor in some
other P450 reactions (51), our demonstration that
b5 serves a role as an allosteric facilitator of
electron transfer from OR to P450c17 was unexpected. The binding of
redox partners to P450c17 must transmit subtle changes to the
substrate-binding pocket, as evidenced by the lower
Km values for
5 substrates in the
presence of human OR (Table III) and by analogy to the altered
regiospecificity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine oxygenation by P450 2D6 in the presence of OR (28). Furthermore, the
oxidative scission of the C17-C20 bond of
17
-hydroxypregnenolone appears to impose much more stringent
constraints on the active-site topology of P450c17 than does the
17
-hydroxylase reaction. Therefore, we propose that
b5 optimizes the geometry of the P450c17·OR
complex for the more sensitive 17,20-lyase reaction perhaps by forming a ternary complex (Fig. 7). The
structural core 2 domain of b5 may be the region
that stimulates 17,20-lyase activity, as core 2 adopts a similar
conformation in the NMR structures of both isolated
holo-b5 (52) and apo-b5
(53), whereas the heme-binding core 1 domain is disordered in
apo-b5 (53). Furthermore, core 2 retains its
overall topology during molecular dynamics simulations of
apo-b5, while core 1 loses secondary structure
and exhibits conformational mobility (54).

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Fig. 7.
Proposed function of cytochrome
b5. I, NADPH donates two electrons
to the FAD domain of OR (touching the microsomal membrane), which then
pass to the FMN moiety. II, the FMN domain of OR, which is
connected to the FAD domain by a connecting domain and a hinge region
(H) must rotate about 90° (counterclockwise in the figure)
to dock with the redox-partner binding site of P450c17. The interaction
of P450c17 and OR is adequate to support 17 -hydroxyation, but this
complex rarely adopts the geometry required to catalyze the 17,20-lyase
reaction. III, the presence of either
holo-b5 or apo-b5 favors
the interaction of OR and P450c17 in an orientation that satisfies the
more stringent conformational restrictions required by the 17,20-lyase
reaction, facilitating productive electron transfer from OR to P450c17
and subsequent catalysis. The precise site(s) of action of
b5 remain unknown.
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A role for b5 as an allosteric effector protein
is consistent with our observation that serine phosphorylation of
P450c17 selectively increases 17,20-lyase activity (8) and that
mutations of arginine residues in the redox-partner binding site of
human P450c17 cause isolated 17,20-lyase deficiency (23). The precise orientation of OR in the electron-donor docking region of P450c17 required to assemble the active oxygenating complex for the 17,20-lyase reaction is impaired by mutation of this surface and enhanced by
b5 or apo-b5.
Phosphorylation of P450c17 probably favors assembly of productive
complexes so that electron transfer is more rapid and coupling
efficiency is higher; however, the exact mechanism by which
phosphorylated serine residues enhance 17,20-lyase activity is not yet
known. A more detailed understanding of these complexes is essential
for understanding the regulation of 17,20-lyase activity; this in turn
may permit development of agents to inhibit this activity, which will
aid in the treatment of sex steroid-dependent malignancies
and disorders of androgen excess.
We thank Dr. Denis Pompon for yeast strains
W(B), W(hR), and W(B
) and for yeast vectors V10 and V60; Drs.
Gregory Petsko and Ira Herskowitz for yeast strains W303A and W303B and
for vector pYcDE2; Dr. C. Roland Wolf for antiserum to human OR; and
Drs. Phillipe Urban and Gilles Truan for valuable discussions.