The Rate-determining Step in P450 C21-catalyzing Reactions in
a Membrane-reconstituted System*
Shiro
Kominami
,
Akiko
Owaki,
Tsuyoshi
Iwanaga,
Hiroko
Tagashira-Ikushiro§, and
Takeshi
Yamazaki
From the Faculty of Integrated Arts and Sciences, Hiroshima
University, 1-7-1 Kagamiyama, Higashihiroshima 739-8521, Japan
Received for publication, July 10, 2000, and in revised form, December 22, 2000
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ABSTRACT |
Adrenal cytochrome P450 C21 in a
membrane-reconstituted system catalyzed 21-hydroxylation of
17
-hydroxyprogesterone at a rate higher than that for progesterone
in the steady state at 37 °C. The rate of product formation in the
steady state increased with the concentration of the complex between
P450 C21 and the reductase in the membranes. The complex formation was
independent of the volume of the reaction, showing that the effective
concentrations of the membrane proteins should be defined with the
volume of the lipid phase. The rates of conversion of progesterone and
17
-hydroxyprogesterone to the product in a single cycle of the P450
C21 reaction were measured with a reaction rapid quenching device. The
first-order rate constant for the conversion of progesterone by P450
C21 was 4.3 ± 0.7 s
1, and that
for 17
-hydroxyprogesterone was 1.8 ± 0.5 s
1 at 37 °C. It was found from the
analysis of kinetic data that the rate-determining step in
21-hydroxylation of progesterone in the steady state was the
dissociation of product from P450 C21, whereas the conversion to
deoxycortisol was the rate-determining step in the reaction of
17
-hydroxyprogesterone. The difference in the rate-determining steps
in the reactions for the two substrates was clearly demonstrated in the
pre-steady-state kinetics.
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INTRODUCTION |
Microsomal cytochrome P450 isozymes are integral membrane proteins
responsible for the metabolism of various exogenous and endogenous
compounds, including steroid hormones. The monooxygenase reaction of
P450 requires two electrons and an oxygen molecule. NADPH-cytochrome
P450 reductase in the microsomal membranes supplies the first and the
second electrons to P450s in the same membrane. Although the supply of
the second electron to P450 is often the rate-limiting step in P450
reactions, results of recent experiments have revealed that some P450
reactions have rate-determining steps other than in the electron supply
(1-5). Bell and Guengerich (1) clearly showed in the oxidation of
ethanol catalyzed by P450
2E11 that the kinetic
deuterium isotope effect on Km with no effect on
kcat was attributed to the rate-limiting product release. P450 2E1 catalyzes the oxidation of ethanol to acetic acid via
acetaldehyde, in which about 90% of the intermediate acetaldehyde is
directly converted to acetic acid without dissociation from the active
site of the enzyme (2). They showed for the first time in P450
reactions that the reactions for two substrates catalyzed by one
molecular species of P450 could have different rate-determining steps
(1). We demonstrated in studies on the successive reactions of P450
11
and P450 17
that the rates of product formation in the steady
state were regulated by the rates of product release from the enzymes
(3-5). P450 17
and P450 11
catalyze multistep reactions for the
formation of androgens and aldosterone, respectively, where the final
products are formed from fractions of the intermediates that do not
dissociate from the enzymes (3-5). The same mechanism for diminishing
the rate of dissociation of final products from P450 2E1, P450 11
,
and P450 17
might decrease the dissociation rates of the
intermediates, facilitating the successive monooxygenase reactions in
these P450s without the intermediate metabolites leaving the enzyme. It
is of interest to examine whether the product release can be the rate-determining step for P450 that does not catalyze multistep reactions.
The interaction between NADPH-cytochrome P450 reductase and P450 has
been investigated by several laboratories in the systems reconstituted
with detergents (6, 7) and dilauroylphosphatidylcholine (8-13). Miwa
et al. (14, 15), using a phospholipid vesicular membrane
system, found that the active species for the P450-catalyzing monooxygenase reaction in the steady state was the binary complex consisting of P450 and the reductase. Kawato and co-workers (16-20) have extensively studied the interaction between P450 and the reductase
in phospholipid vesicles by measuring the rotational diffusion of P450s
in the membranes, and they suggested that the mode of interaction
depends on the individual P450. Rotational diffusion measurement of
P450 C21 in liposomal membranes revealed a complex formation with the
reductase (21). In this study, we obtained experimental evidence that
the effective concentrations of P450 C21 and NADPH-P450 reductase for
the complex formation should be defined with the volume of the lipid
phase of the membranes rather than the total volume of the reaction solution.
Two types of cytochrome P450 function in the endoplasmic reticulum of
the adrenal cortex (22, 23): P450 C21, catalyzing steroid
21-hydroxylation (24, 25), and P450 17
, catalyzing steroid
17
-hydroxylation and androgen formation (26, 27). Some of the
progesterone originally produced from cholesterol is hydroxylated to
17
-hydroxyprogesterone, a part of which is further metabolized to
androstenedione without dissociation from P450 17
(5). Progesterone
and 17
-hydroxyprogesterone are the physiological substrates for P450
C21 and are converted to deoxycorticosterone and deoxycortisol,
respectively. The activity of P450 C21 for the reaction of
17
-hydroxyprogesterone is higher than that for progesterone in
bovine and guinea pig microsomes and also in the reconstituted systems
(23, 24). The difference in the activities of P450 C21 in the reactions
of the two substrates has been investigated by various methods. The
rate of first electron transfer from the reductase to P450 C21 in the
presence of progesterone was not much different from that in the
presence of 17
-hydroxyprogesterone, where the high spin content of
the P450 C21-17
-hydroxyprogesterone complex was higher than that of
the P450 C21-progesterone complex (28). The rates of substrate binding
to P450 C21 in liposomal membranes do not differ much between the two
substrates (29).
To elucidate why the activity of P450 C21 in the steady state is higher
for 17
-hydroxyprogesterone than for progesterone, it is necessary to
determine the rate-determining step in 21-hydroxylation reactions of
progesterone and 17
-hydroxyprogesterone. We performed kinetic
studies on P450 C21-catalyzing hydroxylation reactions of progesterone
and 17
-hydroxyprogesterone in the steady state, in single turnover
experimental conditions, and in the pre-steady state.
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EXPERIMENTAL PROCEDURES |
Preparation of Proteoliposomes--
Cytochrome P450 C21 and
NADPH-cytochrome P450 reductase were purified from bovine
adrenocortical and hepatic microsomes, respectively, according to
methods described previously (24, 30). The purified P450 C21 was
incorporated into unilamellar vesicular membranes by the cholate
dialysis method using phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine at a molar ratio of 5:3:1 (31). P450 C21
proteoliposomes used in this experiment contained about 0.65 nmol of
P450 C21 per mg of the phospholipids, which corresponds to one molecule
of P450 C21 in about 2000 molecules of phospholipids. A certain amount
of the reductase was incorporated in the P450 C21 proteoliposomes by
incubation with the preformed P450 C21 proteoliposomes at 0 °C for
1 h (32). The amount of P450 in the reaction solution was
determined from the dithionite-reduced CO difference spectra (33). In
some cases, the concentration of P450 in the membrane reconstituted
system is expressed in mol of P450/liter of the lipid phase of the
membranes under the assumption that 1 g of phospholipids occupies
1 ml (34).
Assay of 21-Hyroxylase Activity in the Steady State--
The
rates of 21-hydroxylation of P450 C21 proteoliposomes for progesterone
and 17
-hydroxyprogesterone were measured at 37 °C using
3H-labeled substrates (31). In general, 0.5 ml of 50 mM potassium phosphate buffer (pH 7.2) contained 10 pmol of
P450 C21 and various amounts of the reductase (2.5-25 pmol) in the
liposomal membranes (0.015 mg of phospholipids), 10 nmol of
progesterone or 17
-hydroxyprogesterone with corresponding
3H-labeled steroid (0.5 µCi), and 0.1 mM
EDTA. The reaction was initiated by the addition of 100 nmol of NADPH
and terminated by mixing with 1 ml of chloroform containing
14C-labeled androstenedione (0.005 µCi). The
radioactivity of 14C-labeled androstenedione was used for
the estimation of the recovery of steroids in the assay procedures. The
extracted steroids were separated with an HPLC system described
previously (23, 31).
Single Turnover Experiments--
To determine the rate of
conversion from progesterone or 17
-hydroxyprogesterone to the
21-hydroxylated product under single turnover reaction conditions,
rapid quenching experiments were performed at 37 °C with a rapid
quenching device (UNISOKU MX-200) equipped with four cylinders
and two mixers (35). Solutions A and B were mixed rapidly with mixer 1 and stored in a reaction coil. The reacted solution was pushed out from
the coil with solution C and was mixed rapidly in mixer 2 with solution
D, the termination solution. In the single turnover experiment of P450
C21, solution A (100 µl) contained 50 pmol of P450 C21
proteoliposomes with various amounts of the reductase (50-300 pmol)
and 2.5 pmol of the 3H-substrate (0.25 µCi) in 50 mM potassium phosphate buffer (pH 7.2) with 0.1 mM EDTA. Solution B (100 µl) contained 100 nmol of NADPH
and 100 nmol of the unlabeled substrate in the buffer. Solution C (200 µl) was the buffer solution, and solution D (200 µl) was 1 M HCl. Under these experimental conditions, only the reaction of 3H-substrate bound to P450 C21 at the initial
stage of the reaction could be detected. 3H-substrate or
3H-product dissociated from P450 C21 could not be
metabolized again, because of the presence of an excess amount of
unlabeled substrate in the reaction solution. The steroids were
extracted with chloroform after termination of the reaction and
separated by HPLC.
Pre-steady-state Kinetics for P450 C21 Reactions--
The
pre-steady-state kinetics of P450 C21-catalyzing reactions were studied
using a UNISOKU MX-200. Solution A (100 µl) contained 5 pmol of P450
C21 proteoliposomes with 5 pmol of the reductase and 100 pmol of
3H-substrate (1 µCi) in the reaction buffer. Solution B
(100 µl) contained 10 nmol of NADPH but did not contain an unlabeled
substrate. The contents of solutions C and D are the same as those in
the single turnover experiments. In these reaction conditions,
multicycle turnover reactions of P450 C21 can occur. The time courses
of P450 C21-catalyzing reactions of progesterone and
17
-hydroxyprogesterone were measured in the initial 3 s at
37 °C. Computer simulations for the pre-steady-state kinetics were
performed with HopKINSIM version 1.7.2 provided by D. Wachsstock
(Johns Hopkins University) using a Macintosh computer (iMac; Apple
Computer Inc., Cupertino, CA) equipped with FPU 3.07 (John Neil & Associates, Cupertino, CA) (36, 37).
Materials--
[1,2,6,7-3H]progesterone and
[4-14C]androstenedione were obtained from PerkinElmer
Life Sciences. 17
-[3H]Hydroxyprogesterone was produced
from 3H-labeled progesterone by an enzymatic reaction using
P450 17
-proteolipsomes and purified with HPLC (31).
L-
-Phosphatidylcholine from egg yolk was obtained from
Sigma, and L-
-phosphatidylethanolamine from egg yolk and
L-
-phosphatidylserine from bovine spinal cord were from
Lipid Products (Surrey, United Kingdom). Other chemicals were of the
highest grade commercially available.
 |
RESULTS |
Reactions of P450 C21-Proteoliposomes in the Steady State--
The
reaction system of P450 C21 was reconstituted in liposomal membranes.
The incorporation of P450 C21 and NADPH-P450 reductase in the liposome
membranes was confirmed by density gradient centrifugation (31, 32).
Almost all of the P450 C21 in the proteoliposomes is reduced upon the
addition of the reductase and NADPH, suggesting that the heme domain of
P450 C21 interacts with the functional domain of the reductase on the
outer surface of the vesicles. Fig.
1a shows the time courses of
21-hydroxylation of progesterone and 17
-hydroxyprogesterone
catalyzed by P450 C21 at 37 °C in the presence of NADPH-P450
reductase at an equimolar amount of P450 C21 (10 pmol) in the liposome
membranes (15 µg of phospholipids in 0.5 ml of reaction solution).
The amounts of deoxycortisol and deoxycorticosterone, which were the
21-hydroxylated products from 17
-hydroxyprogesterone and
progesterone, respectively, increased linearly with the reaction time.
The rates of product formation by P450 C21, which were calculated from
the slopes of the lines, were 0.33 ± 0.04 and 0.17 ± 0.03 nmol/min for 17
-hydroxyprogesterone and progesterone, respectively.
The rates of product formation both from progesterone and
17
-hydroxyprogesterone were not altered by change in the volume of
the reaction solution as long as the total amounts of P450 C21, the
reductase, and phospholipids were kept constant in the reaction
solution, as shown in Fig. 1b.

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Fig. 1.
Reactions of progesterone to
deoxycorticosterone (open circles) and of
17 -hydroxyprogesterone to deoxycortisol
(open squares) at 37 °C in the steady
state. a, the reactions were carried out in 0.5 ml of
reaction solution with 10 pmol each of P450 C21 and NADPH-cytochrome
P450 reductase and 10 nmol of 3H-labeled steroids (0.5 µCi) in liposome membranes (0.015 mg of phospholipids) for the time
indicated on the x axis. b, the volume of
reaction solution was varied in the range of 0.2-2 ml. The other assay
conditions are the same as in a. The details are described
under "Experimental Procedures."
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Dependence of the Rate of Product Formation on the Amount of the
Reductase in Liposome Membranes in the Steady State--
The rate of
product formation in the steady state increased hyperbolically with the
amount of the reductase in the membranes as shown in Fig.
2, where the rate for
17
-hydroxyprogesterone was almost twice that for progesterone in the
concentration range observed. An increase in the rate of product
formation by various P450s has been observed with the increase in the
amount of the reductase in the reaction solution and has been
attributed to formation of an active complex between P450 and the
reductase (14, 15). The dissociation constant of the active P450
C21-reductase complex (1:1, mol/mol) was estimated by the method of
Miwa et al. (15). We obtained 16 ± 4 and 15 ± 4 nM for Kd(app), the apparent
dissociation constants for the P450 C21-reductase complex, from the
dependence of the rates of 21-hydroxylations of
17
-hydroxyprogesterone and progesterone, respectively. The apparent
dissociation constants were calculated under the assumption that P450
C21 and the reductase are distributed homogeneously in the reaction
solution. These Kd(app) values are a little smaller
than those reported for other P450 systems (14, 38). Below, we discuss
the calculation of dissociation constants of the complex using
effective concentrations of the enzymes defined with the volume of
lipid phase of the membranes. Vmax, which is the
rate in the presence of an excess amount of the reductase with 0.01 nmol of P450 C21, was 0.78 ± 0.06 nmol of deoxycortisol produced
per min for the reaction of 17
-hydroxyprogesterone and 0.48 ± 0.06 nmol of deoxycorticosterone produced per min for progesterone. The
lines in Fig. 2 are the theoretical curves drawn with the above values. It was remarkable that Vmax was
about 2 times higher for the 17
-hydroxyprogesterone reaction than
for the progesterone reaction but that Kd(app) was
about the same for both substrates, suggesting that the difference in
the substrates had little effect on the interaction between P450 C21
and the reductase.

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Fig. 2.
Dependence of the rate of 21-hydroxylation on
the amount of NADPH-cytochrome P450 reductase in liposome
membranes. The reactions of progesterone (open
circles) and 17 -hydroxyprogesterone (open
squares) were measured at 37 °C in the presence of 10 pmol of P450 C21 and various amounts of the reductase. The
lines are theoretical curves drawn using the apparent
dissociation constants of 15 and 16 nM for the P450
C21-reductase complex in the presence of 10 nmol of progesterone and
17 -hydroxyprogesterone, respectively, under the assumption that the
complex between P450 C21 and the reductase (1:1, mol/mol) is an active
molecular species for the reaction. The details are described
under "Results."
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Single Turnover Experiments for P450 C21 Reactions--
To obtain
the rate of conversion of the substrate to the 21-hydroxylated product
in the single turnover reaction, a solution containing a
P450-reductase-3H-substrate ternary complex was mixed
rapidly with excess amounts of NADPH and the unlabeled substrate. Since
3H-substrate and 3H-products once released from
the enzyme do not rebind to P450 C21 in the presence of an excess
amount of unlabeled substrate, only the change in the
3H-substrate in the P450 C21-reductase complex at the
initiation of the reaction is observable under these conditions. Fig.
3, a and b, shows
the time courses of reactions of progesterone and 17
-hydroxyprogesterone, respectively, under the single turnover conditions at 37 °C. The amount of [3H]progesterone
decreased exponentially with the reaction time, and the decrease
corresponded exactly to the increase in 21-hydroxylated 3H-product, [3H]deoxycorticosterone. The
decrease in 17
-[3H]hydroxyprogesterone and the
corresponding increase in [3H]deoxycortisol were
apparently slower than those for progesterone. The first-order rate
constants for reactions of progesterone and 17
-hydroxyprogesterone,
which were obtained by fitting the observed data to single exponential
curves, were 4.3 and 1.8 s
1, respectively. It
is concluded that the higher activity of P450 C21 for the reaction of
17
-hydroxyprogesterone in the steady state is not due to the higher
conversion rate of 17
-hydroxyprogesterone than that of progesterone.
The time courses for reactions of progesterone in the single turnover
conditions were also dependent on the amount of the reductase in the
reaction solution as shown in Fig. 4. The
amount of [3H]progesterone that was converted to
[3H]deoxycorticosterone in the single turnover reaction
increased with the amount of the reductase in the membranes, but the
rate of the conversion did not vary, remaining 4.0 ± 0.5 s
1. The increase in the amount of product in
the single turnover condition could be attributed to the increase in
the amount of active complex in the reaction solution.

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Fig. 3.
Single turnover experiments for the
conversion of progesterone (open circles)
to deoxycorticosterone (closed circles) and
of 17 -hydroxyprogesterone (open
squares) to deoxycortisol (closed
squares), catalyzed by P450 C21-proteoliposomes.
The experiments were performed at 37 °C with a rapid quenching
device (UNISOKU MX-200) in the presence of 50 pmol each of P450 C21 and
the reductase and 2.5 pmol (0.25 µCi) of the 3H-labeled
steroid in the membranes (0.077 mg of phospholipids). The
lines were drawn using the first-order rate constants of 4.3 s 1 and 1.8 s 1 in a
and b, respectively, which were obtained using the
simulation software, Kaleida graph (Version 3.0.5, Albelck
Software). The details are described under "Results."
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Fig. 4.
Effects of the amount of NADPH-cytochrome
P450 reductase on the single turnover experiments for the conversion of
progesterone (open symbols) to
deoxycorticosterone (closed symbols)
catalyzed by P450 C21 proteoliposomes. The experiments were
performed in the presence of a constant amount (50 pmol) of P450 C21
and 50, 100, and 300 pmol of the reductase in liposome membranes,
corresponding to the data points of circles,
squares, and triangles, respectively. Other
experimental conditions are the same as those described in the legend
to Fig. 3. All of the curves are drawn using the first-order
rate constant of 4.0 s 1. The details
are described under "Results."
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Pre-steady-state Kinetics of P450 C21 Reactions--
We measured
the time courses of the 21-hydroxylation reactions at 37 °C under
the pre-steady-state reaction conditions using a rapid mixing device,
UNISOKU MX-200. A solution (100 µl) containing equimolar amounts of
P450 C21 and the reductase (5 pmol) and 100 pmol of radioactive
substrate was mixed rapidly with an equal volume of NADPH solution, and
the conversions of substrates were measured in the range of 0-3 s.
Fig. 5, a and b,
show the conversions of progesterone and 17
-hydroxyprogesterone to
the products, respectively, in which the amount of deoxycorticosterone
increased rapidly in the initial 300 ms with a slower linear increase
after that, while the amount of deoxycortisol increased almost linearly
up to 3 s. The burst increase of deoxycorticosterone in the
pre-steady state showed that the rate of conversion from progesterone
to deoxycorticosterone must be significantly faster than the rate of
dissociation of product deoxycorticosterone from the enzyme (39). The
lines in Figs. 5 are the theoretical curves drawn using the
computer software HopKINSIM (36, 37).

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Fig. 5.
Pre-steady-state kinetics for the reaction of
progesterone (a) and of
17 -hydroxyprogesterone (b)
catalyzed by P450 C21 proteoliposomes. The experiments were
performed at 37 °C in the presence of 5 pmol each of P450 C21 and
the reductase with 100 pmol of substrates (1 µCi) in 200 µl of the
reaction solution using a rapid quenching device. A series of observed
data points are shown with closed circles. The
line in a was drawn with
k1 = 0.02 (nM·s) 1,
k2 = 5 s 1, and
k3 = 1 s 1 and 2.1 pmol
of the active complex using HopKINSIM for the scheme in Fig. 6. The
line in b was drawn with
k1 = 0.02 (nM·s) 1,
k2 = 1.5 s 1, and
k3 = 5 s 1 and 2.1 pmol
of the active complex. The amount of active complex was calculated
using Kd(lip) = 520 µM.
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 |
DISCUSSION |
If P450 C21 and the reductase were distributed homogeneously in
the reaction solution, the effective concentrations of P450 C21 and the
reductase would be decreased to one-tenth by a 10-fold increase in the
volume of the reaction solution. Fig. 1b shows, however,
that there was almost no change in the rate of product formation with
an increase in the volume of the reaction solution, suggesting that the
formation of the active complex does not depend on the volume of the
reaction solution. The concentrations of P450 C21 and the reductase in
the lipid phase of the membranes are not affected by change in the
volume of the reaction solution as long as the absolute amounts of P450
C21, the reductase, and the phospholipids are kept constant. P450 C21
proteoliposomes contain 1 mol of P450 C21 in 2000 × 770 g of
phospholipids, where 770 is the average molecular weight of the
phospholipids. The concentration of P450 C21 was 0.65 mM in
the lipid phase under the assumption that 1 g of phospholipids
occupies 1 ml of the volume in the membranes (34).
The apparent dissociation constants, Kd(app), for
P450 C21-reductase complex were calculated from the reductase
dependence of the rate of product formation under the assumption of a
homogeneous distribution of enzymes in the reaction solution using the
equation Kd(app) = ((P/V)(R/V))/(PR/V),
where P, R, and PR represent the
absolute amounts (mol) of free P450C21, free reductase, and P450
C21-reductase complex, respectively, and V is the volume of
the reaction solution (0.0005 liters in Fig. 2). The amount of P450
C21-reductase complex can be calculated using the equation PR (mol) = (observed rate of product
formation/Vmax) × (total amount of P450
C21 (mol)), where Vmax is the rate when all of the P450 C21 is in the form of a complex with the reductase. The concentration of P450 C21 in the lipid phase can be defined as P/Lip, where Lip represents the volume
in liters of the lipid phase of the membranes. The dissociation
constant of the complex defined in the lipid phase can be written as
Kd(lip) = ((P/Lip)(R/Lip))/(PR/Lip) = Kd(app) × (V/Lip)
(29). In the experiments for Fig. 2 using 10 pmol of P450 C21, the
volume of the lipid phase can be calculated as 10 × 10
12 × 2000 × 770 ml, and V
is 0.5 ml. The value for V/Lip is 3.2 × 104, and the value for Kd(lip) becomes
520 ± 120 µM for P450 C21-reductase complex, which
must be the real dissociation constant for the P450 C21-reductase
complex in the liposome membranes. A similar Kd(lip)
value, about 0.5 mM, is calculated for a hepatic
P450-reductase complex in egg yolk liposomes from the data of Miwa and
Lu (14).
In the experiments for Fig. 4, the amount of P450 C21 was kept constant
(50 pmol in 0.077 mg of phospholipids), and the amount of reductase was
increased from 50 to 100 and then to 300 pmol. The amount of
[3H]progesterone hydroxylated by P450 C21 in the single
turnover reaction increased with the amount of the reductase in the
liposome membranes, but the rate of conversion of
[3H]progesterone did not change.
[3H]Deoxycorticosterone must be produced from the active
ternary complex of P450 C21-[3H]progesterone-reductase
that exists at the initial stage of the reaction. The amounts of active
complex between P450 C21 and the reductase in the lipid phase can be
calculated using the equation Kd(lip) = ((Pt
PR)/Lip)(Rt
PR)/Lip)/(PR/Lip), where Pt and Rt represent the total amounts of P450 C21
and the reductase (mol) in the reaction solution, respectively, and
Lip is 7.7 × 10
8 liters for
the experiments in Fig. 4. The amounts of complex were calculated using
Kd(lip) = 520 µM to be 21, 31, and 43 pmol in the presence of 50, 100, and 300 pmol of the reductase with 50 pmol of P450 C21 in the membranes, respectively. These are
almost proportional to the amounts of product formed in the single
turnover reactions (0.4, 0.55, and 0.7 pmol, respectively) in Fig. 4.
The small amounts of product formation are attributed to the low
concentration of 3H-substrate (2.5 pmol) in the reaction
solution. On the other hand, in the model of P450 C21 and the reductase
being distributed homogeneously in the reaction solution, the amounts
of the complex calculated as 39, 47, and 49.4 pmol using
Kd(app) do not increase significantly with an
increase in the amount of the reductase. Fig. 4 clearly demonstrates
that the effective concentrations of the membrane proteins should be
defined in the lipid phase.
To analyze the reaction mechanism of P450 C21 in the steady state, we
divided one cycle of the P450 C21 reaction into three steps, as shown
in Fig. 6. It is assumed that the complex
between P450 C21 and the reductase is the active species in the
reaction. The presence of an excess amount of the substrate prevents
reactions in the reverse direction. The rate for the conversion of the
substrate to the product obtained in the single turnover experiment
corresponds to k2. The steady-state velocity of
product formation can be written as follows (40),
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(Eq. 1)
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where [E0] represents the concentration
of the active complex. This equation can be simplified as
v/[E0] = k2k3/(k2 + k3) in the presence of an excess amount of
substrate, where
k2k3/k1[S] is much smaller than k2 + k3. We can calculate
the values of k3 for the reactions of progesterone and
17
-hydroxyprogesterone in the steady state using the observed values
of Vmax for v, the rates of
conversion of the substrate, k2, and the total
amount of P450 C21 in the reaction solution. In the presence of an
excess amount of the reductase, nearly all P450 C21 in the reaction
solution must be in the form of an active complex, and the rate of
product formation can be expressed as Vmax. The
calculated values for k3 are listed together
with the values of Vmax and
k2 in Table I. It is quite interesting that
the rate of dissociation of the product from the enzyme,
k3, was about one-fourth less than the rate of
the conversion of the substrate, k2, in the
reaction of progesterone, whereas k3 was about
2.5 times larger than k2 for 17
-hydroxyprogesterone. It is clarified that the rate-determining steps for the reactions of progesterone and 17
-hydroxyprogesterone are the dissociation of the product and the conversion of the substrate, respectively.

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Fig. 6.
Scheme for P450 C21 reaction. A complex
between P450 C21 and NADPH-cytochrome P450 reductase (1:1 mol/mol) was
assumed to be an active molecular species for the hydroxylation
reaction. The reactions in the reverse direction were ignored because
of the presence of an excess amount of substrate steroid. P,
R, S, and D represent P450 C21,
NADPH-P450 reductase, the substrate steroid, and the product,
respectively.
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Table I
Kinetic parameters for P450 C21 reactions in the membrane reconstituted
system
Vmax is the rate of product formation by 10 pmol of
P450 C21 in the presence of an excess amount of the reductase in the
membranes. k2 is the rate constant for the
conversion of the substrate to the product, and k3
is that for the product dissociation from P450 C21-reductase complex in
the scheme shown in Fig. 6. Values are means ± S.D. of at least
triplicate determinations. The details of the calculation are described
under "Discussion."
|
|
The difference in the rate-determining steps in P450 C21 reactions must
affect the pre-steady-state kinetics of the reactions of progesterone
and 17
-hydroxyprogesterone (1). The time courses of product
formation from progesterone and 17
-hydroxyprogesterone in the range
of 0-3 s are shown in Fig. 5, a and b,
respectively. As discussed above, the rate of conversion of
progesterone to the product in the active complex is about 4 times
faster than the dissociation of deoxycorticosterone from the active
complex, which is reflected by the burst and the subsequent slow linear increase of the product. On the other hand, the conversion of 17
-hydroxyprogesterone to deoxycortisol is the slowest step in the
reaction cycle, and we did not observe any burst formation of
deoxycortisol. The lines in Fig. 5 are the simulated curves obtained using the simulation program HopKINSIM version 1.7.2 with the
rate constants in Table I, where the value of k1
was selected to be 0.02 (nM·s)
1
for the binding of both progesterone and 17
-hydroxyprogesterone (29,
36, 37). The simulation study shows that the burst increase in the
product formation is due to the fast conversion of the substrate to the
product with subsequent slow dissociation of the product from the enzyme.
The successive reactions catalyzed by P450 2E1, P450 11
, and P450
17
had as rate-determining steps the dissociation of the products,
with slow dissociation of intermediate products facilitating the
further monooxygenation of intermediates without dissociation from the
enzyme active sites (2-5). The rate-determining step in the
hydroxylation reaction of progesterone catalyzed by P450 C21 is the
product dissociation, and P450 C21 does not catalyze a multistep
reaction. This means that the rate-determining product release from the
enzymes was not restricted to the successive reactions. The
rate-determining product release might not be a special phenomenon in
P450 reactions. White and Coon (41) had speculated some 20 years ago
that the difference in the activity of one species of P450 for
different substrates might be explained by the difference in the
product dissociation rate. The difference in the rates of product
release might be due to the difference in the hydrophobicities of the
products (42, 43). Deoxycortisol has two hydroxyl groups, but
deoxycorticosterone has only one. It is not surprising that
deoxycortisol dissociates faster from P450 C21 than the more
hydrophobic deoxycorticosterone (29). Since it could be the
rate-determining step in the P450 reaction, much more attention should
be paid to the product dissociation from P450s.
 |
FOOTNOTES |
*
This work was supported in part by Grant-in-Aid for
Scientific Research 11116221 on Priority Areas "Biometallics" from
the Ministry of Education, Science, Sports and Culture of Japan.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence and reprint requests should be addressed.
Tel.: 81-824-24-6526; Fax: 81-824-24-0757; E-mail:
kominam@hiroshima-u.ac.jp.
§
Present address: Dept. of Biochemistry, Osaka Medical College, 2-7, Takatsuki, Osaka 569-8686, Japan.
Published, JBC Papers in Press, January 11, 2001, DOI 10.1074/jbc.M006043200
 |
ABBREVIATIONS |
The abbreviations used are:
P450 2E1, cytochrome
P450 having ethanol oxidation activity;
P450 C21, cytochrome P450
having steroid 21-hydroxylase activity;
P450 11
, cytochrome P450
having steroid 11
-hydroxylase activity;
P450 17
, cytochrome P450
having steroid 17
-hydroxylase activity;
HPLC, high performance
liquid chromatography.
 |
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Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
Copyright © 2001 by the American Society for Biochemistry and Molecular Biology.