Kinetic processivity of the two-step oxidations of progesterone and pregnenolone to androgens by human cytochrome P450 17A1

JBC Papers in Press. Published on July 6, 2017 as Manuscript M117.794917

The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M117.794917

Human P450 17A1 Kinetics

Kinetic processivity of the two-step oxidations of progesterone and pregnenolone to androgens by human

S

cytochrome P450 17A1

1

2

Eric Gonzalez and F. Peter Guengerich

From the Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee

7232-0146

3

Running title: Human P450 17A1 Kinetics

Address correspondence to: Prof. F. Peter Guengerich, Department of Biochemistry, Vanderbilt

University School of Medicine, 638B Robinson Research Building, 2200 Pierce Avenue, Nashville,

Tennessee 37232-0146, Telephone:

f.guengerich@vanderbilt.edu

1

(615) 322-2261; FAX:

1

(615) 343-0704; E-mail:

Keywords:

Cytochrome P450, Enzyme kinetics, Pre-steady-state kinetics, Steroid metabolism,

Steady-state kinetics, P450 17A1, CYP17A1

ABSTRACT

human P450 17A1 is an inherently distributive

enzyme but that some processivity is present, i.e.

some of the 17a-OH pregnenolone formed from

pregnenolone did not dissociate from P450 17A1

before conversion to DHEA. Our results also

suggest multiple conformations of P450 17A1, as

previously proposed on the basis of NMR

spectroscopy and X-ray crystallography.

Cytochrome P450 (P450, CYP) 17A1 plays a

critical role in steroid metabolism, catalyzing both

the 17a-hydroxylation of pregnenolone and

progesterone and the subsequent 17a,20-lyase

reactions to form dehydroepiandrosterone (DHEA)

and androstenedione (Andro), respectively, critical

for generating glucocorticoids and androgens.

Human P450 17A1 reaction rates examined are

3

enhanced by the accessory protein cytochrome b

), but the exact role of b in P450 17A1-

5

The cytochrome P450 (P450 or CYP )

(

b

5

enzymes collectively have the most diverse set of

substrates, at least among oxidoreductases (1). In

particular, P450s are very important catalysts in

the metabolism of steroids (human P450s 1B1,

catalyzed reactions is unclear as are several details

of these reactions. Here, we examined in detail the

processivity of the 17a-hydroxylation and lyase

7

2

A1, 7B1, 8B1, 11A1, 11B1, 11B2, 17A1, 19A1,

1A2, 24A1, 27A1, 39A1, 46A1, 51A1, plus some

steps. b

5

did not enhance reaction rates by

decreasing the koff rates of any of the steroids.

Steroid binding to P450 17A1 was more complex

than a simple two-state system. Pre-steady-state

experiments indicated lag phases for Andro

production from progesterone and for DHEA from

pregnenolone, indicating a distributive character of

the enzyme. However, we observed processivity in

pregnenolone/DHEA pulse-chase experiments.

others involved in catabolism) and vitamins

(human P450s 2R1, 24A1, 26A1, 26B1, 26C1,

27A1, 27B1, 27C1) (2, 3). The steroidogenic

P450s are particularly important, and homozygous

or dual heterozygous deficiencies are often

debilitating (2, 4). P450 enzymes also have major

roles in the metabolism of exogenous (xenobiotic)

chemicals, including drugs and carcinogens (3),

and some P450s oxidize both endogenous and

xenobiotic substrates (5).

(

S)-Orteronel was 3 times more inhibitory toward

the conversion of 17a-hydroxypregnenolone to

DHEA than toward the 17a-hydroxylation of

pregnenolone. IC50 values for (S)-orteronel were

identical for blocking DHEA formation from

One of the steroidogenic P450s, P450

7A1, has a critical role in the production of

1

androgens and also glucocorticoids (2). At least 50

variants with P450 17A1 insufficiency have been

identified clinically (4, 6). The enzyme catalyzes

the two-step oxidation of progesterone to 17a-OH

pregnenolone

and

for

17a-hydroxylation,

suggestive of processivity. Global kinetic

modeling helped assign sets of rate constants for

individual or groups of reactions, indicating that

1

Human P450 17A1 Kinetics

progesterone to androstenedione (Andro) and also

the oxidation of pregnenolone to 17a-OH

pregnenolone to dehydroepiandrosterone (DHEA)

but activities were seen in the presence of a 5-fold

molar excess of b

There are several enigmas about P450

17A1. One is the exact role of b , which NMR

> P450 17A1 (12).

5

(

1

Fig. 1A). The second reaction in each series (Fig.

A) is termed a “lyase” (or “desmolase”) reaction,

5

studies indicate occupies the same site as and

competes with the obligate electron donor

NADPH-P450 reductase (13, 22). b does not

5

appear to transfer electrons to P450 at any point in

the catalytic cycle (23, 24). Another controversy is

in that cleavage of the 17,20 C-C bond occurs.

Although these were first considered to be

separate enzymatic activities, they are now known

to be catalyzed by a single protein, P450 17A1 (7-

9

). The presence of another hemoprotein,

whether a ferric peroxide (FeO ¯) or Compound I

2

(perferryl oxygen; FeO ) species is involved in

the lyase reaction (15, 16, 25-27).

3

+

cytochrome b (b ), has been shown to greatly

5

stimulate the lyase activity (10-13), at least in

some species. The process is complicated by

accompanying minor reactions (Fig. 1B). The

minor products have all been identified in vitro

One of the issues regarding the two-step,

net four-electron oxidation (Fig. 1A) is the

processivity of the overall reaction. In a processive

reaction, the 17a-OH steroid product remains

bound to the enzyme for the succeeding lyase

reaction, but in a distributive reaction the 17a-OH

product dissociates and then re-binds for the lyase

reaction (Fig. 1A). A variety of reports on the topic

of processivity have appeared, with varying

conclusions. On the basis of work in human

embryonic kidney (HEK 293) cells, Soucy and

Luu-The (28) concluded that this is a distributive

sequence of reactions. However, studies with rat,

guinea pig, and bovine P450 17A1 enzymes led to

conclusions that the two steps are processive (29-

(

14-16), and 16a-OH progesterone has been

measured in human serum (17).

A role for b in enhancing P450 catalytic

5

activity was first shown in liver microsomes in

1

the 17a-OH progesterone lyase activity of purified

porcine testicular P450 17A1 3- to 4-fold (10, 20).

Kominami et al. (21) also showed stimulation of

guinea pig P450 17A1 by bovine b , both the 17a-

hydroxylation of progesterone and, to

fractionally larger extent, the 17a-OH

progesterone lyase reaction. However, b₅was

971 (18, 19). In 1982 b was shown to stimulate

5

a

3

3). These studies involved

a

number of

inhibitory at a b :P450 17A1 ratio > 0.5 (21). In

1

5

^a^p^p^r^o^a^c^h^e^s^aⁿ^d^d^e^sⁱ^gⁿ^s^b^u^tⁿ^oⁿ^eⁱⁿ^c^l^u^d^e^d^b5^,

995 Katagiri et al. (11) reported that human b₅

which had already been shown to stimulate the

lyase reaction (10, 11). Our own work with

(

added in a concentration equimolar to P450)

stimulated several catalytic activities of human

P450 17A1. Progesterone 17a-hydroxylation was

not stimulated by b₅but pregnenolone 17a-

hydroxylation was enhanced 2.5-fold; lyase

activities toward both 17a-OH progesterone and

zebrafish P450 17A1 and b indicated that the

5

reactions were distributive when progesterone was

the substrate and more processive when

pregnenolone was the substrate, although only a

relatively weak effect of zebrafish or human b is

5

1

7a-OH pregnenolone were enhanced ~ 10-fold

seen in that system (34).

by b (11). No qualitative difference was observed

in the binding of 17a-OH pregnenolone to P450

1

al. (12) also demonstrated stimulation of both

porcine and human P450 17A1 activities by

porcine b , although the results differed from those

of the Waterman group (11). The catalytic

efficiency of 17a-hydroxylation was increased 2-

fold for pregnenolone but not for progesterone, as

in the report of the Waterman group (11).

However, no lyase activity with either 17a-OH

progesterone or 17a-OH pregnenolone (as

substrate) could be detected in the absence of b₅,

5

The matter of processivity is important for

several reasons. One is that 17a-OH progesterone

and 17a-OH pregnenolone (formed following 2-

electron reduction of 17a-OH progesterone) serve

as precursors to glucocorticoids (2, 3). Another

issue is that prostate cancer is androgen-stimulated

7A1. In the same year (1995), Lee-Robichaud et

5

(

35), and P450 17A1 is a major drug target for

therapy (6, 36-39). A goal is the selective

inhibition of the androgen-producing lyase

reaction, preserving the ability to generate 17a-

OH steroids as precursors for biosynthesis of

glucocorticosteroids and avoiding adverse effects

caused by insufficient glucocorticoid and excess

2

Human P450 17A1 Kinetics

mineralocorticoid production. Some selectivity for

lyase > 17a-hydroxylation activity has been

reported for abiraterone (36), orteronel (Takeda

TAK-700) (40), the secosteroid (S)-seviteronel

progesterone (Figs. 2C, 2D). 16a-Hydroxylation

of progesterone was also enhanced by b (Fig. 2C).

5

The major effects of b were on k values (Table

5

cat

1).

(

VT-464) (41), and two other non-steroidal

pyridine derivatives (36). X-ray crystal structures

of human P450 bound to abiraterone (6) and to

both enantiomers of orteronel (42) have been

published by Scott and co-workers. A completely

processive reaction should not have differential

sensitivities to a single inhibitor. Even in a

dissociative mechanism, the question arises as to

how a drug can occupy a single site on the enzyme

Binding of substrates and products to P450 17A1

Initial experiments were done with low

concentrations of P450 17A1 in a 10-cm cell (34)

because of the low K values we estimated in

d

preliminary assays, which were done using the

“Type I” Soret spectral shifts (increase in

absorbance at 390 nm, decrease near 420 nm (49)).

Because of issues we found regarding the

instability of P450 17A1 during the protracted

measurements, we collected data (following

stopped-flow mixing) 7 seconds after additions

and used these results, utilizing a quadratic

and have different K values for two reactions (42,

i

4

3).

For the above reasons, we examined the

kinetics of binding of human P450 17A1 to its

primary physiological substrates and products and

also an enantiomer of orteronel. We also utilized

pre-steady-state reaction kinetics and global data

analysis to develop a model of the human P450

equation for fitting. The apparent K values for

d

pregnenolone, progesterone, 17a-OH progesterone,

and 17a-OH pregnenolone were all sub-

micromolar (Fig. 3). The K value for DHEA was

d

1

7A1 reactions.

1

.7 µM and that for Andro was ~ 19 µM, although

the K fitting for the latter product was not very

accurate.

d

RESULTS

Steady-state reaction kinetics

These studies began with steady-state

reaction kinetics, which included an analysis of the

Measurement of k_o_f_frates of substrates and

products of P450 17A1

One of the possible roles of b in the

5

effect of b (Fig. 2, Table 1). Some of the lack of

5

enhancement of the lyase reactions could be

ideal curve fitting may be due to the formation of

additional products (Fig. 1B) (15, 16), which were

not included here for the sake of simplification.

The efficient conversion of 17a-OH pregnenolone

to DHEA confounds the results for formation of

decreased dissociation of 17a-OH steroids (Fig.

1

A). If this were the case, the k_o_f_frates would be

expected to be lower in the presence of b₅.

Because of the different Soret spectra

observed with various ligands (Figs. 4, 5) and the

interaction of the imidazole group of orteronel

with the heme iron (34), we were able to use this

1

2

7a-OH pregnenolone from pregnenolone (Fig.

A), but the lyase reaction (using 17a-OH

pregnenolone as the starting substrate (Fig. 2B))

was more straightforward. The decrease in

apparent rates of DHEA formation in the presence

compound as a trap (the estimated K for (S)-

orteronel is 40 nM (42), which we confirmed (data

d

not presented)). With 10 µM (S)-orteronel, the

-

1

of b (Fig. 2B) may be the result of facile 16-

apparent on-rate was 6.1 s (Fig. 4). Mixing of

equimolar concentrations (2 µM) of P450 17A1

and each steroid with 10 µM (S)-orteronel

provided a means of estimating k_o_f_fvalues, in that

these were 1-2 orders of magnitude slower.

Spectral data and analysis for 17a-OH

pregnenolone (as an example) are presented in Fig.

5

hydroxylation of DHEA (16). Our k_c_a_tand K_m

values are similar to those of Lee-Robichaud et al.

(

12) and the k_c_a_tvalues are as high or higher than

reported for any human P450 17A1 preparations

by others (11, 44-48).

b₅stimulated all of the reactions examined

-

1

-1

(

Fig. 2). Very little DHEA was formed from

5 (k 0.4 s in that example, 0.32 ± 0.02 s for an

off

pregnenolone or 17a-OH pregnenolone in the

average of more experiments in Table 2).

The trapping method was used to measure

absence of b (Figs. 2A, 2B) nor was very much

5

Andro formed from progesterone or 17a-OH

koff rates for all steroid substrates and products

3

Human P450 17A1 Kinetics

(

Table 2). In no case did b substantially affect k

(Fig. 7B). Even when the chase (unlabeled) 17a-

5

off

values.

OH pregnenolone was added as early as 50

milliseconds, the production of [ H]-DHEA was

3

Time courses of individual P450 17A1 reactions

only blocked by ~ one-third. Collectively these

results indicate a partially processive mechanism

(Fig. 7), which is more pronounced with

pregnenolone oxidation to DHEA than for

progesterone conversion to Andro (Figs. 6B, 6F)).

P450 17A1 reactions were initiated with

equal concentrations (2 µM) of P450 17A1 and

each substrate (Fig. 6). (These experiments are

often termed “single turnover” assays, although

the term is not strictly correct in that substrate

binding and release are occurring on similar time

scales as substrate turnover.) In these assays we

also measured minor products in order to better

account for reaction stoichiometry. Several

Inhibition of P450 17A1 reactions by orteronel

enantiomers

One of the goals in prostate cancer

chemotherapy is attenuation of androgen

production by selective inhibition of the lyase

reaction without disturbing the formation of 17a-

OH products, which are necessary for the

formation of other steroids (2).

apparent

first-order

rates

of

substrate

disappearance were calculated and are presented in

Table 3 (see also Supplemental Data Fig. S1).

All reactions were stimulated by the

presence of b , with the exception of the rate of

5

The (S) enantiomer of orteronel

progesterone disappearance (Figs. 6E, 6F). As

expected, all secondary products showed lags in

formation, including the lyase products DHEA and

Andro (Figs. 6B, 6F).

(

Supplemental Data, Fig. S2) was more inhibitory

than the (R) enantiomer in all reactions (Fig. 8). In

the case of progesterone, both enantiomers were

slightly more inhibitory for the lyase reaction than

1

7a-hydroxylation (Table 4). In the reactions with

Pulse-chase assays with pregnenolone oxidation

to DHEA

pregnenolone, the IC value for the lyase reaction

5

0

was ~ one-third that for 17a-hydroxylation. When

the overall conversion of pregnenolone to DHEA

was analyzed (Fig. 8B), the IC₅₀values (for both

enantiomers) were similar to those measured for

The lag phases for formation of lyase

products (DHEA from pregnenolone, Andro from

progesterone) (Figs. 6B, 6F) are indicative of

distributive mechanisms, in that a completely

processive mechanism should proceed without a

lag. The lag phase was shorter in the case of

pregnenolone (no DHEA appearing until 2

seconds, Fig. 2F) than progesterone (no Andro

appearing until 5 seconds, Fig. 2B), and we

examined this reaction further utilizing pulse-

chase assays (Fig. 7).

1

7a-hydroxylation (Fig. 8A).

Development of kinetic models

For initial model development we used the

measured k_o_f_fand K values to estimate apparent

d

k rates (Table 5). The k rates were not affected

on

by the presence of b (Supplemental Data, Fig.

S3)). We also measured some k rates directly but

these are complex and will be considered

separately (see below, Fig. 11). Only the systems

containing b were considered. The oxidation step

rates are those measured in the single-turnover

reactions presented in Figs. 6B, 6D, 6F, and 6G.

Using the simplified model and values

listed in Fig. 9, it was possible to fit the data using

KinTek Exlorer® software (Fig. 10) for single-

turnover (Fig. 10A, 10B), steady-state (Fig. 10C,

5

3

on

Reactions were initiated with [ H]-

pregnenolone. In steady-state assays (Fig. 7A),

unlabeled 17a-OH pregnenolone was added 60

seconds after the reaction was initiated, and the

reaction proceeded for another 10 min. Even with

adding a concentration of unlabeled 17a-OH

pregnenolone as high as 75 µM, only about one-

5

3

half of the [ H]-DHEA formed from [ H]-

pregnenolone could be blocked.

In pre-steady-state reactions, patterned

after the experiment in Fig. 6B, a chase of 80 µM

unlabeled 17a-OH pregnenolone was added to the

reaction at varying times after the reaction started,

and the reaction proceeded for another 5 seconds

1

0D), and binding reactions (Fig. 10E, 10F, 10G).

As mentioned in the presentation of Fig. 2 (see

above), some of the discrepancy may be due to not

including all secondary products (14-16).

4

Human P450 17A1 Kinetics

We also considered the binding of

pregnenolone, 17a-OH pregnenolone, and DHEA

in detail, using global analysis of the actual kinetic

17A1 to consider the role of b in a processivity

study, which is critical (Figs. 2, 6). The fish

enzyme is only modestly stimulated by fish or

5

binding curves (Fig. 11). The k values used were

human b (34). We conclude that b stimulates

5 5

off

approximately those measured in the trapping

both the human P450 17A1 17a- (and 16a-)

experiments (Table 2). However, the k rates are

hydroxylation and lyase reactions, plus diene

on

slower than estimated from consideration of k_o_f_f

formation (Fig. 6B). b did not affect the affinity of

5

and K values (Table 5). In addition, the estimated

P450 17A1 for 17a-OH steroids (Fig. 3, Table 2).

We believe that multiple conformations of P450

17A1 (Fig. 13) are necessary to explain several of

the kinetic results, in support of NMR

spectroscopic (22) and X-ray crystallographic

evidence (42, 44) for the existence of multiple

conformations of the protein.

d

6

-1

k_o_nvalues are all < 10 M

s

and therefore

probably not diffusion-limited (50).

An alternate binding scheme is proposed,

one in which there exist multiple, interconverting

conformations of P450 17A1 in the absence of

ligands. Independent evidence for the existence of

multiple conformations comes from the NMR X-

ray crystallography and spectroscopy work of

Scott, Laurence, and their colleagues (22, 42, 44).

Using a model in which both the ligand-free and

ligand-bound forms of the enzyme have multiple,

inter-convertible conformations, we were able to

fit the raw data files for binding of pregnenolone,

One previous study concluded that human

P450 was a processive enzyme, but this work was

only done at a cellular level (HEK-293 cells) (28).

Others concluded that the rat (31) and guinea pig

(29) enzymes are processive with the substrate

progesterone, based on microsomal studies.

Another rat microsomal study (30) with

progesterone was unclear regarding conclusions

about processivity but can be interpreted in the

context of a partially processive mechanism.

Tagashira et al. (32) concluded that the guinea pig

enzyme was processive with the substrate

progesterone, and Yamazaki et al. (33) concluded

that the bovine enzyme was “20% processive”; all

of these studies (except those using microsomes)

were done in the absence of b₅.

1

7a-OH pregnenolone, and DHEA to P450 17A1

(

Fig. 12).

DISCUSSION

P450 17A1 plays a critical role as the

immediate enzyme involved in steroid metabolism

following transformation of cholesterol to

pregnenolone by another P450, P450 11A1 (2).

P450 17A1 deficiency is a major endocrinological

issue (3, 4), but the same enzyme is also a current

target in the treatment of prostate cancer (39). In

this paper we investigated several kinetic aspects

of how this enzyme works in the catalysis of two

successive reactions (Fig. 1A). We demonstrate

that this is an inherently distributive process, in

regard to the two successive reactions, with some

of the 17a-OH steroid products dissociating from

the enzyme. However, there is some processivity,

particularly in the case of pregnenolone, i.e., part

of the 17a-OH steroid pool does not dissociate but

remains bound to the enzyme for the second

oxidation step. Accordingly, some of the product

DHEA is derived from 17a-OH pregnenolone that

has not dissociated from the enzyme (Fig. 7). This

is, to our knowledge, the first detailed study to

consider the processivity of isolated human P450

It is clear that any results obtained in the

absence of b are problematic (Fig. 2, Table 1).

5

Lee-Robichaud et al. (12) reported no detectable

lyase activity with either 17a-OH pregnenolone or

1

7a-OH progesterone in the absence of b , but

5

both we (Figs. 2, 6) and others (11, 47) did

observe low activity with human P450 17A1. b₅

has been proposed to play a critical role in the

balance of steroid metabolism, particularly in the

production of androgens (11, 12). One issue is that

the role of b in lyase reactions catalyzed by P450

5

171 varies among species. With zebrafish P450

17A1, the stimulations by (zebrafish) b were only

5

~ two-fold (34). Mice in which b was globally

5

deleted were still viable and reproduced, even

though the formation of androgens from

progesterone and 17a-OH progesterone in testis

microsomes was severely attenuated and the (in

vivo) intratesticular levels of testosterone were

reduced by one-half (51). In vitro (murine)

1

7A1 and, with the exception of our zebrafish

P450 17A work (34), the first of any with P450

5

Human P450 17A1 Kinetics

microsomal 17a-hydroxylation of progesterone

was also reduced ~ one-half. Again, there is

species variability and the application to humans is

not direct. In humans, some clinical endocrine

disorders involving low levels of androgens have

complex by b

5

is not involved in the stimulation of

P450 17A1 catalytic activity (23, 24) is

complemented by our more recent findings that

P450 17A1 activity supported by the oxygen

surrogate iodosylbenzene, in the absence of

NADPH-P450 reductase in the system, is also

been linked to defective variants of b (52, 53). In

5

the two cases cited (52, 53), the (in vivo) levels of

augmented by b (16).

5

The stimulatory effects of b (Table 1, Fig.

) are clearly not related to differential effects on

binding of intermediate products (Table 2). The

1

7a-OH steroids were not compromised.

5

2

P450 17A1 is primarily an adrenal enzyme,

and one question is what the relative levels of b₅

and P450s are there. Some information is available,

but the situation is complex in that there are four

zones in the adrenal gland—glomerulosa,

fasciculate, reticularis, and medulla. To our

knowledge, the levels of the relevant enzymes

have not been measured in human adrenal tissue.

Hamamoto et al. (54) quantitated several enzymes

in bovine adrenals using immunochemical

methods. P450 17A1 was not measured but

another microsomal P450 (21A2) and two

mitochondrial P450s (11A1, 11B1) were. The

effects of b on the lyase reactions with both

5

pregnenolone and progesterone were seen in

single-turnover experiments (Fig. 6C, 6D, 6G, 6H).

What is less clear is why the effect of b on 17a-

5

hydroxylation in the steady-state kinetic assays

(Fig. 2, Table 1) is much greater than seen in the

single turnover studies (Fig. 6). Recently, Peng et

al. (47) provided evidence for improvement of

electron coupling of P450 17A1 by b , but

5

presumably the same factors would influence

abortive oxygen formed in the steady state and

under pre-steady-state conditions. We do not have

levels of b were lower than any of the three P450s

5

in all four zones (54). This situation contrasts with

liver, where the microsomal concentrations of

a clear definition of exactly what the kcat or K

m

parameters mean in this complex system, although

the stimulation of both 17a-hydroxylation

reactions is quite clear (Fig. 2A, 2C).

(

total) P450 and b are similar (55).

5

In modeling our results, we focused on

grouping several individual steps together, in order

to simplify the system. Our work indicates that

substrate binding is more complex than a 1-step/2-

state system (Fig. 12), but we modeled the system

accordingly to satisfy several data sets (Figs. 9,

b₅is still an enigma in terms of its role in

the P450 17A1 system. As already pointed out,

there is considerable evidence against a role for

electron transfer, i.e. catalytic activity in assays

with the heme deleted from b (23) or replaced by

5

1

0). In a similar way, we know that each oxidation

Mn-substituted heme (24) and with the oxygen

sequence (either 17a-hydroxylation or the lyase

step) done by P450 17A1 probably involves at

least nine distinct steps (56, 57). We have modeled

each of these sets with a single rate or rate

constant (Figs. 9, 11), without trying to discern

rate-limiting steps (there is evidence that C-H

bond breaking is at least partially rate-limiting in

the 17a-hydroxylation step (14)).

surrogate iodosylbenzene (16).

publication by Duggal et al. (48) argues for

electron transfer from b based on negative results

of experiments with Mn -substituted b

contrast to others (24). However, the rate of lyase

activity with the control b was only ~ 4% of that

measured here (Table 1, Fig. 2) or elsewhere (16)

A

recent

5

2

+

, in

5

(

^s^e^e^a^l^s^o^o^t^h^e^r^r^a^t^e^s⁽¹¹^,¹²^,⁴⁷⁾⁾^,^aⁿ^d^a^c^oⁿ^t^r^o^l^b5

experiment with reconstituted Fe-substituted heme

^w^a^sⁿ^o^tⁱⁿ^c^l^u^d^e^d^.^T^h^e^w^eⁱ^g^h^t^o^f^e^vⁱ^d^eⁿ^c^eⁱ^s^t^h^a^t^b5

b₅showed stimulation of all of the P450

1

7A1 reactions (Fig. 2). In some previous work

induces conformational changes in P450 17A1

the conclusion was reached that b selectively

5

(

and probably several other P450s in which it

enhanced the lyase reaction, possibly by

plays a non-redox role (59) in facilitating product

formation (13)), possibly through more productive

coupling (47). Exactly how this occurs is unclear.

stabilizing the ferric peroxide (FeO ¯) form of the

enzyme proposed to be selectively involved in the

lyase reaction (12, 58). Our findings that multiple

2

There is NMR evidence that b occupies the same

reactions are stimulated by b argues against this

5

site of P450 17A1 that NADPH-P450 reductase

does (22). An enigma is that this evidence

conclusion (Fig. 2, Table 1). Previous evidence

that electron transfer to P450 or its oxygenated

6

Human P450 17A1 Kinetics

suggests that the P450 17A1-iron oxygen complex, explain preferential inhibition of individual

whatever it may be, receives all of its electrons

and is in an activated state before the reductase

reactions by any drug. Alternatively, a second

enzyme site can exist and might be involved (42).

leaves and is replaced by b , which induces a more

A

potential issue related to the

5

favorable conformation for catalysis. This process

must happen rapidly, occurring in every reaction

processivity of P450 17A1 is application to

discovery and development of drugs (for prostate

cancer) that would only inhibit the lyase reaction,

in order to avoid side effects. A number of reports

of selectivity for the lyase reaction > 17a-

hydroxylation have appeared (40-42, 61, 62).

However, repeated assays with purified P450

17A1/reductase/b₅systems have yielded less

selectivity, and some of the reports of high

selectivity have not been confirmed (36, 42). Our

own results with orteronel show IC values (Table

cycle. Assuming the above conclusion that b and

5

NADPH-P450 reductase compete for the same

P450 17A1 site and cannot be bound

simultaneously (22), it is possible that b could

5

bind earlier but it would have to be released for

2

+

NADPH-P450 reductase to rebind (to the Fe ·O

2

complex) for electron delivery—and then rebind.

We previously reported that b could stimulate the

5

lyase activity of P450 17A1 supported by the

oxygen surrogate iodosylbenzene (16).

5

0

4) similar to those reported very recently by

Petrunak et al. (42), although we did not observe

an 11-fold selectivity for (R)-orteronel. Some of

the discrepancies among IC values are attributed

A

role for multiple P450 17A1

conformations, indicated by NMR spectroscopy

13) and X-ray crystallography (42, 44), is

(

5

0

supported by some of our own results. As

indicated, we were unable to fit the substrate

binding data with only a simple 2-state system

to the use of different substrate concentrations,

which necessarily affect IC₅₀estimates. Some

reported selectivity comparisons are not directly

related to processivity, in that 17a-hydroxylation

of progesterone has been compared with lyase

activity towards 17a-OH pregnenolone (36). In

some cases, more selectivity has been reported in

cell cultures, based only on measurement of

endogenous steroids (36, 40, 62), although the

reasons are not clear.

(

Fig. 12). We propose that not only the ground

state form of P450 17A1 but also the substrate-

bound form (13, 42, 44) and probably several of

the electronic intermediates in the reaction

pathway have multiple conformers (Fig. 13). Such

a diagram is consistent with current general

thoughts about enzyme mechanisms (60) and is

probably operative here (Fig. 13). Attempts to

expand our kinetic analysis to include extra

conformations with KinTek Explorer® were

unsuccessful due to the complexity of the system,

and even if more rate constants could be attached

to the system (in Fig. 13) we do not have enough

data constraints to judge the validity of any

modeling that might fit. Therefore our kinetic

conclusions about processivity rely on models in

which reaction steps are grouped together (Figs. 9,

Two findings of the recent Petrunak et al.

report (42) are particularly important: (i) the two

different protein conformations for binding the (R)

and (S) enantiomers of orteronel, and (ii) extra

binding space near the F/G helix region only in the

case of the (S)-orteronel:P450 17A1 complex. The

finding of multiple (or at least two) conformations

of ligand-bound P450 17A1 is consistent with the

complexity of some of our kinetic data (Fig. 12).

Another point is that if there is a second,

peripheral ligand binding site in P450 17A1, then

it may be possible to have an induced fit

component (63) instead of—or in addition to—a

conformational selection model (Fig. 13) for P450

17A1. In principle, the second site of ligand

binding could possibly be involved in generating

reaction-specific inhibition with drugs targeting

P450 17A1.

1

1). The nature of multiple conformations

proposed in the scheme in Fig. 13 is unknown.

These could be only structural conformers.

However, these could also be b -bound and non-

5

b -bound forms (13, 22), reductase and non-

5

3

+

reductase-bound forms, FeO ¯ and FeO forms

2

poised to do different reactions (a possibility we

have not ruled out (16)), or forms that

preferentially bind orteronel instead of the

substrate and product. With an enzyme in which

there appears to be only one ligand binding site,

multiple conformations (Fig. 13) are required to

An alternate consideration for achieving

reaction-specific inhibition of P450 17A1 is

targeting residues that bind b (needed for efficient

5

7

Human P450 17A1 Kinetics

1

7a-OH pregnenolone lyase activity) (Fig. 2B,

Table 1). An issue here is that one would need to

identify (b binding) sites that are not needed to

“distributive” mode, and the composite would be

the results we observe here. The results have

relevance for the development of prostate cancer

treatment drugs that selectively inhibit the

individual reactions.

5

bind NADPH-P450 reductase (22). One approach

would be developing drugs to bind residues (e.g.,

Glu-305, Arg-347, Arg-358, Arg-449) where

natural mutations attenuate lyase activity but not

EXPERIMENTAL PROCEDURES

1

3

7a-hydroxylation activity (6, 64). Arg-347, Arg-

58, and Arg-449 were identified as being

Reagents

involved in the interaction of P450 17A1 with b

using NMR spectroscopy (22), and Glu-305 is

located in the canonical active site (6).

In conclusion, we have analyzed several

kinetic aspects of catalysis by human P450 17A1,

particularly the coupling of the two major steps,

5

Most of the steroids were obtained from

either Sigma-Aldrich or Steraloids (Wilton, NH).

DHEA was purchased from Waterstonetech

3

(Carmel, IN). [7- H(N)]-pregnenolone was

purchased from PerkinElmer (catalog number

1

4

NET039001MC).

ARC1398)

[4- C]-Progesterone

3

(

and

17α-OH-[1,2,6,7- H]-

1

7a-hydroxylation and the lyase step, with both

progesterone (ART 0638) were purchased from

American Radiolabeled Chemicals (St. Louis,

the substrates progesterone and pregnenolone. b₅

has an ancillary role in several reactions, and some

possible roles were ruled out (e.g., enhancing

binding of 17a-OH steroids). The individual

sequential reactions are inherently distributive but

more processive in the case of pregnenolone than

with progesterone (e.g., Fig. 6B, 6F lags). There is

some processivity, especially with pregnenolone,

as demonstrated by the pulse-chase experiments

MO). The (S)-orteronel enantiomer was

generous gift of Millennium Pharmaceuticals

Cambridge, MA).

Radiolabeled 17a-OH pregnenolone was

a

(

prepared by enzyme-mediated conversion of [7-

3

H(N)]-pregnenolone (65), with modification.

7a-OH-[7- H(N)]-pregnenolone was obtained

3

1

from a 30-second incubation with the enzyme

(

Fig. 7). Our kinetic model has 17a-OH

system described below, devoid of b . The

pregnenolone undergo a 1:6 partitioning between

proceeding to the lyase reaction and dissociation

from P450 17A1 (Figs. 9, 11). In addition to the

pulse-chase data, another point of evidence in

favor of some processivity of the 17a-

hydroxylation and lyase reactions is the orteronel

inhibition results with pregnenolone (Table 3).

With both enantiomers of orteronel, the lyase

reaction was ~ 3-fold more sensitive to orteronel,

confirming the results reported by Hara et al. (40)

and Petrunak et al. (42) with (S)-orteronel. When

5

radiolabeled steroid was purified by the

3

chromatographic method used for H-17a-OH

pregnenolone assays, and fractions were collected

(

^wⁱ^t^h^t^h^e^β^-^R^A^M^s^y^s^t^e^m^oⁿ^l^y^u^s^e^d^t^o^l^o^c^a^t^e^t^h^e^tR

of the product, disconnected for the preparative

mode). The steroid was extracted from the mobile

phase with CH Cl , the organic (lower) layer was

2

transferred to a new vessel, and the solvent was

evaporated under a nitrogen stream. The dried

extract was dissolved in C H OH, and co-elution

2

5

with commercial 17a-OH pregnenolone was used

to further validate the identity of the radiolabeled

compound.

DHEA

production

was

measured

from

pregnenolone, the IC₅₀value was similar to the

value for the 17a-hydroxylation reaction, not the

lyase reaction (measured beginning with 17a-OH

pregnenolone) (Table 3). This result is consistent

with the view that a part of the P450 17A1

proceeds through the reaction without its 17a-OH

pregnenolone being available for exchange (to

allow orteronel to enter the active site). In

considering the hypothetical scheme presented in

Fig. 13, it is conceivable that one or more

conformational form(s) of the enzyme might be in

a “processive” mode while others are in a

Purification of (R)-orteronel

A racemic orteronel mixture was purchased from

ApexBio (Houston, TX) (catalog number A4326),

3

and the compound was dissolved in CH OH for

purification of the enantiomers. The (S) and (R)

enantiomers were resolved on a Chiralcel® OJ-RH

column (4.6 mm ´ 150 mm) with an isocratic

6

3.5% CH

3 2

OH/36.5% H O mobile phase (v/v),

with baseline resolution. The two enantiomeric

8

Human P450 17A1 Kinetics

fractions with absorbance peaks at 238 nm were

collected (Supplemental Data, Fig. S2). The (S)

and (R) fractions were identified using a standard

mM potassium phosphate buffer (pH 7.4). The

P450 17A1 enzyme concentrations used were:

0.01 µM for incubations with pregnenolone and

17α-OH pregnenolone, 0.01 µM and 0.5 µM for

progesterone 17a-hydroxylation and 16a-

hydroxylation (respectively), and 0.5 µM in 17a-

OH progesterone lyase assays. Substrate was

(

S)-orteronel sample, which eluted first. (R)-

Orteronel was extracted from the aqueous solution

with CH Cl and the solvent was evaporated in

vacuo. The dried solid was stored at -20 °C until

2

further use. The two enantiomers had identical UV, added at concentrations ranging from 0.2 to 20

NMR, and mass spectra, except for a slight

difference in the chemical shifts attributed to the

µM. Duplicate samples were pre-warmed at 37 °C

for 5 min (water bath with shaking) and reactions

were initiated with the addition of an NADPH-

generating system (10 mM glucose 6-phosphate,

0.5 mM NADP , and 2 µg/ml yeast glucose 6-

phosphate dehydrogenase) (68). The mixtures

were incubated for 5 min at 37 °C. Reactions were

solvent (CD

UV (H O) e238 98 mM cm , e273 13 mM cm ;

NMR (400 MHz, CD OH) d 2.98 (s, 3H, CH ),

.10 (m, 4H, pyrrolidinol methylenes), 7.65-8.00

m, 6H, naphthyl protons), 8.10 and 8.35 (s, 2H,

imidazole ring protons) (solvent impurities at d

.19 and 3.6, C OH; d 1.25, hexanes; d 3.31

CH OH; d 4.87, H O); high resolution mass

spectrum: calc. for C18

R) m/z , (S): m/z , fragmentation of either yielded

dominant ions at m/z 280 (loss of 18, i.e. loss of

O) and m/z 186 (naphthyl methylcarboxamide,

loss of pyrrolidinolpyrrole entity)); o optical

3

OH). (Supplemental Data , Fig. S2);

-

1

-1

2

+

3

(

2 2

quenched with 2 ml of CH Cl , mixed with a

vortex device, and placed on ice. The samples

were centrifuged to separate the organic and

aqueous layers, and 1.8 ml of the organic phase

was transferred to a new vessel and evaporated to

dryness under a nitrogen stream. The extracts from

1

2 5

H

3

2

+

18 3 2

H N O (MH ) 308.1394,

(

progesterone

incubations were dissolved in 100 µl of a CH

and

17a-OH

progesterone

CN-

H

2

3

2

4 °C

H O (1:1, v/v) mixture, and 10 µl aliquots were

2

rotation (S): [a]

c 0.03, CH OH) (compare to +83.8 (61))

sufficient (R)-orteronel was not obtained to

D

+96 ± 6 (n= 3 readings)

analyzed by UPLC-UV. The pregnenolone and

(

3

1

7a-OH pregnenolone incubation extracts were

dissolved in 50 µl CH OH, mixed with 200 µl of

3

accurately measure [a]).

ꢀ

Enzymes

cholesterol oxidase (0.5 units/reaction) in 50 mM

potassium phosphate buffer (pH 7.4) (14, 34), and

incubated at 30 °C (with shaking at 200 rpm) for

Escherichia coli recombinant human P450

7A1 was prepared as described (16). E. coli

recombinant rat NADPH-P450 reductase and

human b were prepared as described by Hanna et

6

-12 hours. The extraction procedure was then

1

repeated, and the samples were redissolved and

analyzed as progesterone, 17a-OH progesterone,

and Andro (derived from pregnenolone, 17a-OH

pregnenolone, and DHEA, respectively). The

reaction products were resolved on an Acquity

BEH C18 UPLC octadecylsilane column (2.1 mm

5

al. (66) and Guengerich (67), respectively.

Cholesterol oxidase (from Streptomyces sp.) was

purchased from Sigma-Aldrich (catalog number

C8649).

´

100 mm, 1.7 µm) with mobile phases A (70%

CH OH/30% H O, v/v) and B (CH CN), at a 0.2

ml/min flow rate. The mobile phase linear gradient

proceeded as follows: 0-1 min, 95% A; 4 min,

3

2

3

Catalytic assays

Steady-state incubations

7

1

0% A; 4.5 min, 60% A; 4.55-6.75 min, 5% A; 7-

0 min, 95% A (all v/v). The column temperature

The steady-state catalytic assays were

generally conducted using a reconstituted enzyme

system in a final reaction volume of 0.5 ml. The

reaction mixtures typically contained 0.01-0.5 µM

human P450 17A1, 2 µM rat NADPH-P450

was maintained at 40 °C, and the sample chamber

was held at 4 °C. The reaction products were

identified by co-elution with commercial standards

and quantified by the A243 peak areas. kcat and K

m

reductase, 0.5 µM human b

5

(when included), and

values were estimated using non-linear regression

of hyperbolic fits in Prism software (GraphPad,

San Diego, CA).

1

6

µM L-a-1,2-dilauroyl-sn-glycero-3-

phosphocholine (DLPC) (added as lipid vesicles

after sonication of a 1 mg/ml aqueous stock) in 50

9

Human P450 17A1 Kinetics

OH pregnenolone, and DHEA) and

A

243

Single-turnover conditions

(progesterone, 17a-OH progesterone, 16α-OH

progesterone, and Andro)) and quantified by

radiochromatogram peak areas using the β-RAM

software (Laura , LabLogic Systems, Brandon,

FL).

Single-turnover

incubations

were

performed using an RQF-3 quench-flow

instrument (KinTek Corp, Snow Shoe, PA). The

instrument operates through a series of rapid

mixing steps through two in-line incubation

chambers, where each step dilutes the samples

two-fold. The instrument temperature was

conditioned at 37 °C. The enzyme substrate

mixture in these reactions included 4 µM steroid, 4

µM human P450 17A1, 8 µM rat NADPH-P450

TM

Pulse-chase assays

Pulse-chase experiments were performed

3

to follow the two-step conversion of [ H]-

3

pregnenolone to [ H]-DHEA under steady-state

and single-turnover conditions. The reconstituted

enzyme-substrate mixture used in the steady-state

pulse-chase assays was the same as for the

protocol described for steady-state incubations,

with the following alterations: (i) 0.5 µM human

5

reductase, 4 µM human b (when noted), and 32

µM DLPC (lipid vesicles) in 100 mM potassium

phosphate buffer (pH 7.4). The following

radiolabeled substrates were used in these studies:

3

[

H]-pregnenolone (10 µCi/µmol), [ H]-17a-OH

P450 17A1, and (ii) 50 µM [ H]-pregnenolone (13

1

4

pregnenolone (700 µCi/µmol), [ C]-progesterone

µCi/µmol). One minute after the reactions were

started with the NADPH-generating system,

unlabeled 17a-OH pregnenolone was added to the

mixtures, at varying concentrations (5-75 µM

(final concentration) and vehicle), and the

incubations continued for ten minutes before

quenching with HCl (0.67 M final concentration).

The mixtures were centrifuged to clear

precipitated protein and the steroid products were

resolved and quantified by the HPLC methods

employed in the single-turnover experiments (see

above), with the following linear gradient: 0-9

min, 75% B; 13.4 min, 86% B; 13.5-15 min, 90%

B; 16-25 min, 75% B (all v/v).

3

(

60 µCi/µmol), and [ H]-17a-OH progesterone

3

10 µCi/µmol). The reactions were initiated by

rapid mixing with 1 mM NADPH in 50 mM

potassium phosphate buffer (7.4) in the first

reaction chamber. The samples were incubated

from 0.5 to 30 seconds, at which time the reactions

were quenched with HCl (1 M) as they were

pushed through the second reaction into the

collection vessel. Four time point replicates were

collected into the same vessel for analysis to

compensate for the low specific activity of [ C]-

progesterone, and the procedure was applied for

the other substrates. The samples were centrifuged

at 2,000 ´ g for 5 min to eliminate precipitated

protein, and the supernatant was directly injected

for HPLC-UV-in-line liquid scintillation counting.

The reaction products were resolved on a Zorbax

RX-C8 octylsilane column (4.6 mm ´ 250 mm, 5

µm) with mobile phase solvent mixtures A (95%

1

4

The pulse-chase assays done with single-

turnover conditions followed a protocol similar to

that outlined above, with some differences. The

standard single-turnover procedure included one

reaction phase, while a pulse-chase experiment

requires two incubation periods. This was

achieved in the rapid quench instrument by

utilizing the second reaction chamber for

incubating with a chase compound, with the

quenching agent placed in the collection vial. The

reagent concentrations were increased so that,

after two dilutions, the working concentrations in

the chase step (second phase) matched those from

the single-turnover incubations. The specific

H

2

O/5% CH

3 3

OH, v/v) and B (95% CH OH/5%

2

O, v/v). With the column at ambient

temperature, the following linear gradients (1

3

14

ml/min) were used: [ H]-pregnenolone, [ C]-

3

progesterone, and [ H]-17a-OH progesterone

assays: 0-10 min: 70% B; 15-18 min, 100% B; 20-

3

0 min, 70% B; [ H]-17a-OH pregnenolone

assays: 0-15 min, 65% B, 20-23 min, 100% B, 25-

5 min, 65% B (all v/v). The scintillation fluid

3

activity of [ H]-pregnenolone was adjusted to 830

3

TM

(

Liquiscint , National Diagnostics, Atlanta, GA)

μCi/μmol, and 80 μM unlabeled 17a-OH

pregnenolone was used as the chase compound.

The pulse length was varied between 0.05 and 2 s

and the chase continued for 5 seconds before the

flow rate was 3 ml/min. The reaction products

were identified by co-elution with commercial

standards (monitored at A216 (pregnenolone, 17a-

1

0

Human P450 17A1 Kinetics

reaction was quenched in the collection vessel

with HCl (0.6 M final concentration). The

mixtures were centrifuged to clear precipitated

protein, and the steroid products were resolved and

quantitated by the method described for the

steady-state pulse-chase assays.

Steroid binding was reanalyzed, with fresh

enzyme samples for each steroid concentration,

using a stopped-flow instrument equipped with a

20-mm cell and a rapid scanning monochrometer

(OLIS RSM-1000, On-Line Instrument Systems).

The stopped-flow sample syringes were

conditioned at 37 °C and filled with either 2 μM

human P450 17A1 or 0.2-20 μM steroid in 100

mM potassium phosphate buffer (7.4) with 120

μM DLPC (lipid vesicles). After mixing, the

instrument recorded the absorbance spectra from

350-500 nm at one millisecond intervals for up to

7 seconds. The differential absorbance maxima

and minima were determined by subtracting the

final and initial (0 seconds) spectra. The composite

absorbance changes, Δ (Amax – Amin), from three

replicates were averaged and plotted against the

ligand concentrations. The data were fit with a

quadratic binding formula to estimate binding

Orteronel inhibition

Competitive inhibition studies with the

orteronel enantiomers were performed and

analyzed by the methods described for steady-state

assays. The inhibitors were added to the enzyme

substrate mixture with the final composition as:

0

.005-100 µM (S)- or (R)-orteronel, 5 µM

substrate, 0.01 µM human P450 17A1 (0.1 µM in

assays with 17a-OH progesterone), 0.5 µM human

b , 2 µM NADPH-P450 reductase, and 16 µM

5

DLPC (lipid vesicles) in 50 mM potassium

phosphate buffer (pH 7.4). In order to resolve an

inconsistent orteronel migration profile, the mobile

constants (K ) using GraphPad Prism, with the

d

2

phase solvents were changed to

NH CH CO /70% CH OH/30% H O for A and 5

mM NH CH CO /90% CH CN/10% H O for B

all v/v). IC50 values were estimated in GraphPad

5

mM

equation Y=B+(A/2)(1/E)((K +E+X)-((K +E+X) -

d

1

/2

(4EX))) .

4

3

2

3

2

4

3

2

3

2

(

Inhibitor trapping

Prism using the Onesite-Fit logIC50 formula:

Y=bottom+(top-bottom)/(1+10^(X-log IC50)).

Spectral enzyme-inhibitor trapping assays

were used to measure dissociation rates for the

steroid ligands. The analysis is dependent upon a

faster inhibitor binding rate versus the dissociation

rates for the steroids. The experiments were

performed at ambient temperature using the

stopped-flow apparatus described above and (S)-

orteronel as the trapping inhibitor. The on-rate for

(S)-orteronel was first measured by mixing 4 µM

human P450 17A1 in 100 mM potassium

phosphate buffer (7.4) (with 120 µM DLPC (lipid

vesicles)) and 20 µM (S)-orteronel (in the same

buffer). The rate was estimated by singular value

decomposition (SVD) analysis of the experimental

data matrix with the following parameters:

absorbance: measured value, wavelength: 350-500

nm (200 points), and time: 0-1 second (one scan

per millisecond). The data was fit with a two-

species sequential model (simple first-order) and

the calculated rates from at least three replicates

were averaged. To estimate the dissociation rates

(k ), the steroid ligands (4 µM) and human b (4

Binding Studies

Ligand binding

Initial binding studies were performed

using previously reported methods (34). In a 10-

cm cell (Starna Cells, Atascadero, CA, catalog no.

3

4Q-100, 25 ml) and using an OLIS-Cary 14

spectrophotometer (On-Line Instrument Systems,

Bogart, GA), the steroid ligands were

incrementally added to 0.1 µM human P450 17A1

in 50 mM potassium phosphate buffer (pH 7.4).

Experiments were conducted as a series of

additions, and the enzyme samples were exposed

to ambient temperature, with mixing and exposure

to a strong light beam (OLIS-Cary14 instrument)

for periods > 60 min. Subsequent control

experiments, where ligand was not added,

exhibited spectral instability in the enzyme over

the same period of time. Specifically, a time-

dependent decrease in the Soret band was

observed, which is problematic considering that

the change in the Soret band (upon ligand binding)

is the outcome analyzed in the assays (no obvious

precipitation occurred).

off

5

µM, when included) were added to the enzyme

solution. The time component was 0-10 seconds

(averaged mode, 62 scans per second) and data

were analyzed as stated.

1

Human P450 17A1 Kinetics

Kinetic analysis

Steroid association rate (kon) parameters

were calculated from the corresponding

dissociation rates (koff), estimated from the

inhibitor trapping assays, and the dissociation

the “activating reagent” was fixed at 1 μM and a

-1

8

K of 1 μM was set with k

= 100 μM s (10

eq

-1 -1

forward

-1

M s ) and k

= 100 s , with the intention of

reverse

forcing the step to be rapidly reversible in order to

prevent any kinetic influence on the other

constants (K

d

), derived from the steroid binding

parameters.

Additionally,

the

reversibility

experiments, using the equation:

accounted for uncoupling (loss of iron-oxygen

K = k / k

species to reactive oxygen species or H O,

d

off

on

2

3

+

The experimental data from the catalytic

assays, under single-turnover and steady-state

conditions, and steroid titrations were concurrently

fit to a minimal kinetic model using KinTek

ExplorerÒ software (KinTek, Snow Shoe, PA).

The predetermined rate parameters (k , k , K ,

and single-turnover substrate conversion rates)

were used as starting values in the model, and in

some cases, the values were fixed. The limits for

the molar extinction coefficients that were applied

to the spectral binding experiments were derived

from the prior fitting with a quadratic formula in

GraphPad Prism (see above). The imported data

sets included the averaged data points with

standard deviation (when applicable).

resulting in Fe ) that can occur during Compound

I (or ferric peroxide) formation. Any rate

limitations derived from uncoupling were

considered to be factored into the rate of the

chemical reaction step. Simulations without the

activation step produced similar results with the

same constraints.

on

off

d

Steroid binding kinetics was evaluated by

applying different enzyme-ligand binding models

to the kinetic data obtained in stopped-flow

binding assays (see Ligand binding) using KinTek

ExplorerÒ software. The data were reduced by

taking the difference of the kinetic traces (~7

seconds trace, 1 scan per millisecond) at the

wavelengths previously designated for A_m_a_xand

A_m_i_n(e.g. A₃₉₁and A₄₂₆kinetic traces were

subtracted for pregnenolone assays; see Ligand

binding and Figs. 5, 12). Three replicate difference

traces for each steroid concentration were

averaged and then normalized to t = 0. The kinetic

traces were then reduced once more by boxed

averaging (n = 5) with standard deviation before

import into the KinTekÒ software.

In order to accurately fit the ligand

titration and catalytic experiments in a single

model,

a

“kinetically silent” second order

activation step was added to prevent the software

from regarding the binding assay as a catalytic

experiment. The activation step corresponds to the

combined P450 17A1 reaction steps that lead to

the chemically competent iron-oxygen species (i.e.

reduction by NADPH-P450 reductase, binding of

molecular oxygen, protonation, and loss of water),

which were not evaluated. The concentration of

Acknowledgements—We thank M. V. Martin, L. M. Folkmann, L. D. Nagy, and T. T. N. Phan for

preparing NADPH-P450 reductase and b and K. Trisler for assistance in preparation of the manuscript.

5

We also thank T. T. N. Phan and K. M. Johnson for acquiring spectra of the orteronel enantiomers and P.

L. Polavarapu for the use of the polarimeter.

Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this

article.

Author contributions—E.G. purified the enzymes, did the kinetic analyses, analyzed the data, and did

the model fitting. F.P.G. conceived the experimental plan, participated in some of the stopped-flow

kinetic studies, analyzed the data, and reviewed all results. E. G. and F. P. G. both wrote the manuscript.

This manuscript constitutes part of the Ph. D. thesis of E. G. at Vanderbilt University.

1

2

Human P450 17A1 Kinetics

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Human P450 17A1 Kinetics

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FOOTNOTES

*

This work was supported in whole or in part by National Institutes of Health Grants R01 GM118122

(

F.P.G.), R01 GM103937 (F.P.G.), and T32 ES007028 (for E.G.). The content is solely the responsibility

of the authors and does not necessarily represent the official views of the National Institutes of Health.

The authors declare that they have no conflicts of interest with the contents of this article.

S

This article contains supplemental Figs. S1-S3.

1

Current address: National Center for Advancing Translational Sciences (NCATS), National Institutes of

Health, Bethesda, MD 20892.

2

To whom correspondence should be addressed: Dept. of Biochemistry, Vanderbilt University School of

Medicine, 638B Robinson Research Bldg., 2200 Pierce Ave., Nashville, TN 37232-0146. Tel.: 615-322-

2

261; FAX: 615-343-0704; E-mail: f.guengerich@vanderbilt.edu.

3

3+

The abbreviations used are: Andro, androstenedione; b , cytochrome b ; Compound I, formal FeO

5

oxidized form of a hemoprotein; DHEA, dehydroepiandrosterone; DLPC, L-a-1,2-dilauoryl-sn-glycero-

-phosphocholine; OH, hydroxy; P450 (or CYP), cytochrome P450; SVD, singular value decomposition.

3

1

7

Human P450 17A1 Kinetics

a

Table 1. Steady-state parameters for human P450 17A1 reactions

-

1

-1 -1

k /K , µM s

K , µM

k , s

m

cat

m

+

/- b₅

-b₅

+b₅

-b₅

+b₅

-b₅

+b₅

Substrate

Reaction

ratio

Pregneno- 17α-

lone Hydroxylation

0.0037

0.11

0.5

±0.3

b

1.1

0.0074

0.10

14

±

0.0005 ±0.01

±0.2 ±0.0044 ±0.02

b

Lyase

0.045

0.49

0.092

±

0.003

±0.14

±0.027

1

7α-OH

pregnenoloneLyase

0.0059 0.071

0.57

0.91

0.010

0.078

8

4

±

0.0012 ±0.005 ±0.46

±0.24 ±0.009 ±0.021

1

7α-

Progesterone Hydroxylation

0.086

0.17

9.7

4.3

0.0089 0.040

±

0.004 ±0.01

±0.8

±0.4 ±0.0009 ±0.004

1

6α-

Hydroxylation

0.0064 0.025

1.1

5.3

0.0058 0.0047 0.8

±

0.0009 ±0.003

±0.6

±1.3 ±0.0030 ±0.0012

1

7α-OH

progesterone Lyase

0.00031 0.019

2.9

7.1

0.00011 0.0027

25

±

0.00001 ±0.001

±0.3

±0.3 ±0.00001 ±0.0001

a

See Fig. 2 for plots. The S.E. values for k and K are calculated from within each experiment,

cat

m

as estimated using Prism non-linear regression analysis. In terms of comparing k values to most

cat

-

1

literature rates for this enzyme, be aware that most have been expressed in units of min . These

-

1

are expressed here in units of s in order to link these to other reaction rates considered in this

work.

b

The rate was too low to accurately measure the steady-state kinetic parameters (see Fig. 6A

below),

1

8

Human P450 17A1 Kinetics

Table 2. k rates estimated by singular value decomposition (SVD) analysis

off

-

1

k , s

off

-b₅

+b₅

Pregnenolone

0.23

0.32

0.80

±

0.02

0.05

0.29

0.41

0.77

±

0.03

0.05

1

7α-OH pregnenolone

DHEA

Progesterone

0.46

0.81

1.8

±

0.02

0.08

0.2

0.42

0.92

1.4

±

0.03

0.04

0.1

1

7α-OH progesterone

Andro

Results are presented as means ± S.D. for at least triplicate determinations.

1

9

Human P450 17A1 Kinetics

a

Table 3.

Rates of substrate disappearance in single-turnover reactions

-

1

Substrate

Presence of b₅

Rate, s

Pregnenolone

—

+

0.28 ± 0.01

0.43 ± 0.02

1

7a-OH pregnenolone

—

0.0074 ± 0.0006

+

—

+

0.77 ± 0.01

Progesterone

0.46 ± 0.02

0.47 ± 0.02

1

7a-OH progesterone

—

0.0066 ± 0.0005

+

0.029 ± 0.001

a

Values are from the experiments in Fig. 6. All values are first-order and include the S. E. values

calculated from curve fitting. See Supplemental Data Fig. S1 for plots and fitting.

2

0

Human P450 17A1 Kinetics

Table 4. IC values for inhibition of steroid oxidation by orteronel enantiomers

5

0

a

IC (95% CI), μM

5

0

Substrate

Pregnenolone

Product

7α-OH

(S)-Orteronel

(R)-Orteronel

1

pregnenolone

1.5 (1.1-1.9)

1.4 (1.2-1.8)

6.4 (4.7-8.6)

6.3 (4.0-9.7)

DHEA

1

7α-OH

pregnenolone

DHEA

0.49 (0.22-1.1)

0.32 (0.22-0.48)

2.8 (1.1-7)

1

7α-OH

Progesterone

progesterone

2.1 (1.6-2.8)

1.3 (0.86-2.0)

1

6α-OH

0

.23 (0.15-0.35)

progesterone

1

7α-OH

progesterone

Andro

0.23 (0.17-0.31)

1.5 (1.0-2.3)

a

See data in Fig. 8. Fitting was done in GraphPad Prism (Onesite-Fit logIC50).

2

1

Human P450 17A1 Kinetics

Table 5. k rates calculated from K and k

off

on

d

-

1

-1

k_o_n, μM s

b₅

0.62

-

+b₅

0.78

0.79

Pregnenolone

1

7α-OH

0

.62

pregnenolone

DHEA

0.47

0.98

0.45

0.89

Progesterone

1

7α-OH progesterone

0.85

0.97

Andro

0.095

0.074

See Fig. 3 and Table 1 for K and k values. See also Supplemental Data Fig. S3.

d

off

2

Human P450 17A1 Kinetics

Fig. 1

A

O

7

O

1

8

21

O

12

20

16

OH

1

19

13

1

4

2

9

1

4

15

10

8

7

3

O

5

O

6

Progesterone

17 -OH Progesterone

Androstenedione

O

OH

HO

17 -OH Pregnenolone

HO

Pregnenolone

DHEA

O

B

OH

O

^C^H3

O

OH

Androstenedione

O

CH₃

O

21

CH₃

CH

3

OH

O

1

7

1

6

O

^C^H3

O

6

OH

O

OH

Progesterone

O

CH OH

2

O

OH

O

^C^H3

CH₃

HO

OH

DHEA

17

O

16

O

CH₃

OH

CH₃

OH

HO

Pregnenolone

HO

OH

HO

Figure 1. Oxidations catalyzed by P450 17A1. A, major classical reactions; B, expanded repertoire of

reactions (14-16).

2

3

Human P450 17A1 Kinetics

Fig. 2

8

6

4

2

.0

5.0

B

5

DHEA (+b )

A

17α-OH preg (+

b

5

)

4

3

.0

DHEA (+

b

5

)

2.0

1

.0

5

DHEA (-b )

5

17α-OH preg ( -b )

0

5

10

15

20

0

5

10

15

20

[Pregnenolone], µM

[17α-OH pregnenolone], µM

1

0.0

1.0

0.8

0.6

0.4

0.2

0

C

17α-OH prog (+

b

5

)

5

Andro (+b )

D

8.0

6.0

4.0

2.0

0

5

17α-OH prog ( -b )

1

6α-OH prog (+

5

b )

16α-OH prog ( -b )

5

Andro (-b )

0

5

10

15

20

0

5

10

15

20

[Progesterone], µM

[17α-OH progesterone], µM

Figure 2.

Steady-state kinetics of major reactions catalyzed by P450 17A1 and the effect of b₅.

The data points are means of duplicate assays (± range). Fitting was done by non-linear regression

analysis of hyperbolic data in GraphPad Prism. See Table 1 for calculated values. A, pregnenolone; B,

1

7a-OH pregnenolone; C, progesterone; D, 17a-OH progesterone.

2

4

Human P450 17A1 Kinetics

Fig. 3

0

.

2

5

0

0.

2

0

5

0

A

B

D

2

0

.

1

15

.1

.10

.05

0

.0

5

0

2

4

6

8

10

0

2

4

6

8

10

[

Pre

g

nenolo

n

e

],

μ

M

[Prog

e

sterone],μM

0

.15

0.15

C

.

10

05

0

0.10

.

0.0

5

0

2

4

6

8

1

0

2

4

6

8

10

[

17α-OH p

r

egneno

l

o

ne

]

, μM

[17α-OH p

r

ogeste

r

o

ne

]

, μM

0

.

1

0

2

0

8

6

0

.

0

5

4

3

2

1

0

E

F

.0

.04

.0

2

0

2

4

6

8

1

0

2

4

6

8

10

[

DHEA],

μM

[Andro], μM

Figure 3.

Binding of substrates and products to P450 17A1. Absorbance measurements were

made following mixing in a stopped-flow spectrophotometer as described in detail in the Experimental

Procedures section. At least three data points were collected at each ligand concentration, and the points

were fit to hyperbolic curves using Prism software and non-linear regression analysis (S.E. calculated

from curve fitting). A, pregnenolone (K 0.37 ± 0.03 µM); B, progesterone (K 0.47 ± 0.04 µM); C, 17a-

d

OH pregnenolone (K 0.52 ± 0.10 µM); D, 17a-OH progesterone (K 0.95 ± 0.08 µM); E, DHEA (K 1.7

d

±

0.2 µM); F, Andro (K 19 ± 9 µM).

d

2

5

Human P450 17A1 Kinetics

Fig. 4

1

0

.

0

8

1

0

.0

.8

B

A

Species 1

Species 2

.

.6

.4

.2

.

6

4

2

0.

0

.

0

3

0

3

50

37

5

40

0

425

45

0

4

7

5

00

5

0

3

7

5

400

42

5

4

5

0

475

500

Wavelength, nm

1

0

.

4

2

1.

3

1

0.0

4

.02

.00

C

1

.3

0

D

E

.

Eigenv

ector

.29

0

.0

.8

Ex

p

er

imental Data

Spec

i

es 1

s 2

to

xperimental

1.28

Spec

e

1

.

2

7

6

5

4

3

.6

Eigenvec r

E

Data

.4

-

0.02

.2

0

-

0

.04

0

0.2

0

.4

0.6

0.8

1.0

0

.2

0

.4

0

.6

0

.8

1.0

0

.2

0

.4

0

.6

0

.8

1.0

Time, s

Figure 4.

(S)-Orteronel binding to P450 17A1. P450 17A1 (2 µM) and (S)-orteronel (10 µM) in

1

00 mM potassium phosphate buffer (pH 7.4) were mixed together in the stopped-flow spectrophotometer.

A, series of spectra collected at 1 millisecond intervals over a total period of 1.0 second (only a subset is

shown). B, calculated beginning and ending spectra after SVD analysis using a 2-species model in the

OLIS GlobalWorksÒ software. C, SVD analysis of data for disappearance of starting species and

appearance of final species, along with OLIS EV (Eigenvector) absorbance and OLIS normalized

experimental data. D, Eigenvector (solid line) and normalized experimental data. E, residuals analysis of

-

1

fit to a rate of 6.1 s (from parts C and D).

2

6

Human P450 17A1 Kinetics

Fig. 5

1

0

.0

.8

.6

.4

.2

1.0

0.8

0.6

0.4

0.2

0

A

B

Species 1

Species 2

0

3

50

375

400

425

450

475

500

350

375

400

425

450

475

500

Wavelength, nm

1

0

.5

.0

.5

1.435

0.010

C

D

Eigenvector

E

1

.425

Experimental Data 0.005

Species 1

Species 2

Eigenvector

Experimental Data

1.415

0

1

.405

-0.005

-0.010

0

1.395

0

1

2

3

4

5

6

7

8

9 10

0

1

2

3

4

5

6

7

8

9

10

0

1

2

3

4

5

6

7

8

9 10

Time, s

Figure 5.

Estimation of k rate for 17 -OH pregnenolone from P450 17A1 using (S)-orteronel

a

off

trapping. The experiment of Fig. 4C was repeated except that the P450 17A1 in the starting system was

bound in a 1:1 molar complex with 17a-OH pregnenolone and the scan was extended to 10 seconds using

the averaging mode (62 scans/second). Under these conditions (4 µM P450 17A1 (E) and 4 µM 17a-OH

pregnenolone (S) and using the K value of 0.52 µM (Fig. 3), 70% of the P450 17A1 is in the substrate-

d

-

1

bound form (K = [E] [S] /[ES]). The calculated k rate was 0.4 s (part D, with the residuals shown

d

free

off

in part E). This is a single experiment; for the entire set of averaged experiments see Table 2.

2

7

Human P450 17A1 Kinetics

Fig. 6

-b₅

+b₅

2

1

0

.0

.5

.0

.5

0

2.0

1.5

1.0

0.5

Pregnenolone

17α-OHpreg

Pregnenolone

A

B

DHEA

Diene

17α-OHpreg

1

6α,17α-(OH) preg

2

DHEA

16α-OH DHEA

16α,17α-(OH) preg

2

0

5

10

15

20

25

30

0

5

10

15

20

25

30

2

1

0

.0

.5

.0

.5

2.0

C

17α-OHpregnenolone

D

17α-OHpregnenolone

1.5

1.0

0.5

DHEA

16α,17α-(OH) preg

2

16α-OH DHEA

DHEA

1

6α,17α-(OH) preg

2

0

5

10

20

25

30

5

10

15

20

25

30

2

1

0

.0

2.0

Progesterone

16α-OHprog

Progesterone

17α-OHprog

E

F

Andro

.5

.0

.5

1.5

1.0

0.5

17α-OHprog

16α,17α-(OH) prog

2

16α-OHprog

1

6α,17α-(OH) prog

2

0

5

10

20

25

30

5

10

15

20

25

30

2

1

0

.0

2.0

17α-OHprogesterone

H

G

7α-OHprogesterone

1

.5

.0

.5

0

1.5

1.0

0.5

0

Andro

1

6α,17α-(OH) prog

2

Andro

6

β,16α,17α-(OH) prog

3

1

6α,17α-(OH) prog

2

6

β,16α,17α-(OH) prog

3

5

10

15

20

25

30

5

10

15

20

25

30

Time, s

Figure 6.

substrates. In each case a mixture of 4 µM P450 17A1, 8 µM NADPH-P450 reductase, 32 µM DLPC,

.10 M potassium phosphate buffer (pH 7.4), and (when indicated) 4 µM b were mixed with an equal

“Single-turnover” kinetics of 1:1 molar complexes of P450 17A1 with various

0

5

volume of the 0.10 M potassium phosphate buffer (pH 7.4) containing 1 mM NADPH to initiate reaction

2

8

Human P450 17A1 Kinetics

in a KinTek RP-3 rapid quench apparatus. At each indicated time point, the reaction was terminated and

the products and residual substrate were separated and quantitated by radio-HPLC as described under

3

14

3

Experimental Procedures. In each case, H- or C-labeled substrate was used ([ H]-pregnenolone (10

3

14

3

µCi/μmol), [ H]-17a-OH pregnenolone (700 µCi/μmol), [ C]-progesterone (60 µCi/μmol), or [ H]-17a-

OH progesterone (10 µCi/μmol)). Each point is derived from a single time-point analysis. Parts A, C, E,

3

and G did not include b ; parts B, D, F, and H included b . Traces are shown for reactions in which the

5

substrate was: A and B, pregnenolone; C and D, 17a-OH pregnenolone; E and F, progesterone; G and H,

7a-OH progesterone. See Supplemental Data Fig. S2 and Table 3 for calculated rates of substrate

disappearance.

1

2

9

Human P450 17A1 Kinetics

Fig. 7

1

000

A

8

6

4

2

00

0

10

20

30

40

50

60

70

80

[17α-OH pregnenolone], µM

2

5

0

5

0

5

0

B

2

1

pulse (s)

chase (s)

0.05

5

0.5

5

1

5

2

5

7

-

Figure 7.

Pulse-chase assays of conversion of pregnenolone to DHEA. A, steady-state reaction.

A reaction was initiated by mixing P450 17A1 (0.5 µM), NADPH-P450 reductase (2 µM), b (0.5 µM),

5

3

DLPC (16 µM), and [ H]-pregnenolone (13 µCi/µmol, in 50 mM potassium phosphate buffer) with an

NADPH-generating system (68). After 60 seconds, the indicated concentration of unlabeled 17a-OH

pregnenolone was added and the reaction was continued for another 10 minutes, at which time the

3

0

Article Doi

Kinetic processivity of the two-step oxidations of progesterone and pregnenolone to androgens by human cytochrome P450 17A1

DOI: 10.1074/jbc.M117.794917

Source and publish data:

Authors:

Article abstract of DOI:10.1074/jbc.M117.794917

Full text of DOI:10.1074/jbc.M117.794917

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