Testicular 17β-hydroxysteroid dehydrogenase: Molecular properties and reaction mechanism

REVIEW

A STEROIDS

A

STEROIDSREVIEW

Hiroshi Inano and’ Bun-ichi

Tamaoki

TESTICULAR 17/3HYDROXYSTEROID DEHYDROGENASE: _MOLECLT,AR PROPERTIES AXD REACTION MECELWISM

STEROIDS

48/l-2

July-August

1986 (l-26)

TESTICULAR 17,3-HYDROXYSTEROID DEHYDROGENASE: MOLECULAR PROPERTIES AND REACTION MECHANISM * Hiroshi

Inano at and Bun-ichi

Tamaoki

b

aNational Institute of Radiological Sciences 9-1, Anagawa-4-chome, Chiba-shi 260, and b Faculty of Pharmaceutical Sciences, Nagasaki University, l-14, Bunkyo-machi, Nagasaki-shi 852, Japan Received

IJovemhe= 3, 1986 INTRODUCTION

17B-Hydroxysteroid NADP+

dehydrogenase

17-oxidoreductase,

important because

and in

the

role

A4-pathway,

androstenedione

by

this

enzyme,

is

androstene-36,178-diol

which

more

(Figure

androgenic

enedione

than

in

while

is

by

is in

as

produced

5-

converted

to

reduction

potentiation

10 times

of androst-

could

of

from

to

is about

dehydrogenase

an

A5-pathway,

enzyme

subsequently

androstenedione,

plays

formation,

the

this

1). As testosterone

regarded

testis

testosterone

converted

by 17B-hydroxysteroid

crinologically

in

testosterone

dehydroepiandrosterone

testosterone

1.1.1.64)

EC

essential

(17P-hydroxysteroid:

its

be endo-

biological

activity. 17D-hydroxysteroid

Histochemically,

detected

been abundantly cells

of the testis

method genase, were

in Leydig

porcine

(1). Recently,

using an antibody almost

testis

STEROIDS

all cells

in interstitial

of

against the

located

in the

has

or Leydig

by an immunocytochemical

dehydrogenase

July-August

tissue

17B-hydroxysteroid

(2). Biochemically,

48/l-2

dehydrogenase

molecules

interstitial after

1986 (l-26)

dehydro-

manual

stained

tissue

of

separation

3

4

Inanoand Tamioki

JId?Lko& (bf

(a) t

4.

2

fd)

Figure 1. Function of 17P-hydroxysteroid dehydrogenase. 1: 178-Hydroxysteroid dehydrogenase. 2: A'3@-Hydroxysteroid dehydrogenase coupled with A5-A'isomerase. (al Androstenedione. (b) Testosterone. Cc) Dehydroepiandrosterone. Cd)5-Androstene-3P,178diol. of the interstitial tissue from the seminiferous tubules of rat testes, 178-hydroxysteroid dehydrogenase activity per unit weight of protein in the interstitial tissue was 44 times higher than that of the seminiferous tubules (3).

The

organelle and cytosol fractions were prepared by a conventional differential centrifugation from rat testicular homogenates, and then the organelle confirmed steroid

under

an

dehydrogenase

the microsomal

electron was

fraction

was morphologically

microscope.

found

to

be

The

170-hydroxy-

localized

m.ainly in

fraction.

Furthermore, by a discontinuous sucrose density centrifugation of the microsomal fraction In the

testicular

rough-

and

microsomal

smooth-surfaced

fraction microsomal

the presence was

divided

fractions,

of CsCl,

into the which

are

17/MWDROXYSTEROiDDEHYDROGENASE

respectively reticula. found

to

be

being

concentrated

indicating

from

ticulum

from granular

178-Hydroxgsteroid

fraction, tion

derived

that

the

androstenedione

of the located

dehydrogenase

the

is

distributed

ovary

and

paper,

we

review

the

properties

of

in

organs

molecular

several

animals. of

and compare

17D--hydroxysteroid

Besides

kidney,

properties

dehydrogenase,

:j.

re-

17R-hydroxysteroid

placenta,

in

produc-

endoplasmic

cells

zland,

was

microsomal

of testosterone

agranular

testicular

other

17!3-hydroxysteroid

site

interstitial

in

skin,

activity

smooth-surfaced

is the

testicular

and acr,ranularendoplasmic

dehydro%enase

in the

5

liver, In this

testicular

them with

dehydroqenase

in

the

other

organs.

PURIFICATION

OF THE ENZY6?E

AND ITS WJLTIFUNCTION Rat several of

physical

to

we were

able

Red

porcine

procedures

dehydrosenase,

was

subjected

to

for solubilization

but

the

enzyme

was

to the biomembrane

15). In 1974,

to solubilize

17B-hydroxysteroid

dehydroqenase

testicular

microsomal

an 1830-fold

increase

fraction

bound

of 3.3% after

cion

and chemical

be tightly

and achieved

not

microsomal

17D-hydroxysteroid

found

from

testicular

membrane

purification

six different siqificantly

HE3B testes

steos even

chromatography showed

of pie; by

sonication,

with an overall

(6). The overall

yield did

by agarose-immobilized

.7/. The enzyme

20a--hydroxysteroid

yield

Pro.

purified

from

dehydrosenase

:EC

6

Inano and Tamioki

1.1.1.149) activity to the extent of one-seventh of that of 17B-hydroxysteroid

dehydrogenase.

h5-38-hydroxy-

Neither

steroid dehydrogenase (EC 1.1.1.145) nor alcohol dehydrogenase

(EC 1.1.1.1) activity was detected

in the purified

enzyme preparation (6). Bogovich and Payne (8) purified the rat

testicular

178-hydroxysteroid

dehgdrogenase

with

an

overall yield of 11% after solubilization with 0.8% sodium cholate in 1M XC1. However, purified 17B-hydroxysteroid dehydrogenase from rat testes seemed to be very labile, because the greatest loss of enzyme activity occurred during the purification procedures (9) or after storage for 2 weeks at -5O'C (8). The predominant activity in human term placenta preferentially Engel

oxidizes

estradiol-17'$ to

(10) specifically named

the

estrone.

enzyme,

Ryan

and

estradiol-17D

dehydrogenase (EC 1.1.1.62). The placental enzyme was concentrated in the cytosol fraction obtained from the homogenates

and was stabilized by a relatively high concentra-

tion of glycerol. Purification of the placental enzyme to homogeneity was achieved through four relatively easy steps with an overall recovery of 74%. The procedure indicated an approximately 500-fold purification from the ammonium sulfate precipitate stage, and 3,000-fold purification from the homogenates (11). A constant ratio of estradiol-17D dehydrogenase activity to 20a-hydroxysteroid dehydrogenase activity was observed during the purification from human placenta by affinity chromatography with agarose-immobilized estrioi-lb-

17/34YDROXYSTEROIDDEHYDROGENASE hemisuccinate-3-methyl

'7

ether. The purified preparation was

apparently homogeneous by

SDS-polyacrylamide

gel electro-

phoresis and possessed the biochemical characteristics reported for estradiol-170 tion

of

the

dehydrogenase.

estradiol-176

This

dehydrogenase

and

co-purifica2Oa-hydroxy.-

steroid dehydrogenase supports the hypothesis that a single enzyme mediates both the catalytic reactions (12). On the other hand, estradiol-170 dehydrogenase was localized in the microsomal fraction of mare's placenta

and was solubilized

in 1.5% sodium cholate. Subsequent purification was achieved by

two

agarose

affinity and

chromatographies

using reactive

estriol-16-hemisuccinate-Sepharose.

blue

2-

Estradiol-

I_70 dehydrogenase has been purified up to 15,000-fold with an apparent recovery of CA 100%

(13). Sheep ovarian 179'-

hydroxysteroid dehydrogenase was purified about l,OOO-fold from the homogenates, using affinity chromatography on estrone-aminocaproate-Sepharose (14). By application of affinity chromatography on Procion Red HE3B coupled with agarose, + which has been shown to have a higher affinity for NADP dependent

enzyme

than

for NAD+-dependent

one

(151,

17R-

hydroxysteroid dehydrogenase was separated from 17a-hydroxysteroid dehydrogenase (EC 1.1.1.148) in hepatic cytosol of female rabbit and purified ;16). l.7P-Hydroxysteroiddehydrogenase was solubilized by hemolysis of rat erythrocytes, and the

purified

enzyme

was

isolated

from the membrane-free

hemolysate by affinity chromatography on Sepharose-immobilized Cibacron Blue F3G-A (17).

8

Inano and Tamioki

iJOLECULAR WEIGHT OF TiiEE;gZY:/IE AND ITS HETEROGENEITY Molecular steroid

weight

of the porcine

dehydrogenase

was estimated

acrylamide

gel

electrophoresis 17)

electrophoresis Molecular

weight

dehydrogenase chromatography weight

over

a

androstenedione the enzyme tosterone the

coefficient purified was

two

the

isoelectric

that of

as

a

of the

the

for

amount

their of

form of

amount

of

and

amino

acidic

a

over

sedimentation 19).

35,930

by

II

The

which

6S3S-polyacrylinto for

at p1

least 4.'?, by

The two molecules acid

amino

amino acids per molecule,

tes-

through

dehydrozenase,

in slucrose gradient. in

active

178-hgdroxysteroid

wei,ght

5.5

contained

testosterone

separable

71

centrifuged

which

with

homogeneous was

17$-hydroxy--

sedimentation

monomer

molecular

I

identical

basic

of

(6).

molecular

was

of the

distribution

apparently

focusing

and

enzyme

gradient,

form

gel

filtration

the

testicular

170-hydroxysteroid

enzyme

gel

by neasurin.g the

electrophoresis,

almost l),

the

from

estimate

formed during

and

testicular

gel

(Table

density

disc

173-hydroxgsteroid

183,000 to

by

by gel filtration

purified

active

3.11s

as

34,000

:4ADPH. Nigration

occurred

proteins,

were

sucrose

From

confirmed

amide

order

the

enzymatically

dehydrogenase

1: 1'3) ,

testicular

as

was determined

gradient,

36,500

form of porcine

and

gradient.

the

In

,31.

de!lydrogenase,

10°C

rat

estimated

of the active

steroid at

the

17P-hydroxy-

by SDS-poly--

as

and 33,200-35,500

of

was

testicular

in

composition

acids

exceeded

agreement

with

17/HWDROXYSTEROlDDEHYDROGENASE

9

Table 1 Amino acid composition of porcine testicular 17i3hydroxysteroid dehydrogenase and human placental estradiol-178 dehydrogenase Amino acid residues 17!3-Hydroxysteroid Estradiol-170 dehydrogenase dehydrogenase Amino acid Enzyme Ia Enzyme IIa North Americanb French' Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan

19

5 17 26 16 18 34 % 19 2 24 5 12 30 85 2

Total residues 286 Mol. weight 36,500 a, ref. 18. b, ref. 53. c, ref. 54.

17 6 14 25 15 17 31 22 24 20 2 21 5 11

20 14 44 42

29

82 12 26 2

;: 16 24 4

632 67,000

620-628 68,000

34-;: 54-56 32-34

;; 56

6&i

2; 72 12 64

t8" 54 12 52 12

:

2 2

274 34,700 ---

the acidic isoelectric points

30

__--.

(18). Furthermore,

by

--

iso-

electric focusing in polyacrylamide gel, the enzyme I was separated into two isozymes (~1 5.5 and 5.6), and enzyme II was also separated into two ip1 4.9 and 5.0). The heterogeneity

of

the

porcine

testicular

174-hydroxysteroid

de-

10

Inane

and Tamioki

hydrogenase charge

was

due

to

the

presence

a

different

net

in molecular

size

of

on the molectile, but not difference

(7).

were

Charge

isomers

detected

also

of

in

the

female

rabbit

kidney

(Mr=31,200-35,100)

50,000-54,000) The

But,

weight (22).

nase dissociated

gel

native

of

three

isoelectric

The

in

prominent without

determined

of

and

existence

of three

which

the

same

molecular

weight

interact sition

the

monomers

to form six dimers of

human on

determined there were

placental

North

from the

from two genetically

in

On the

bands

obtained

a by are

monomers,

a different

net

amounts

can

unequal

,23). When the amino acid corngoestradiol-178

American

some serious

ences result

existin;

focusin?

yielded

different

with

ultra--

dehydroqe-

faint

The results

the

and

by

81.1urea.

two

with

charge,

molecular

9;el electrophoresis.

consistent have

to a

dehydrogenase

urea.

'9 , .r=

from human

estradiol-17D

bands

pi5

endometrium

purified

presence

estradiol-176

focusing

guinea

bands by isoelectric

the

from

p;el electrophoresis.

was

purified

three protein

yolyacrylamide

pattern

human

band, corresponding

of 67,700

obtained

(161,

by SDS-polyacrylamide

a molecular

hand,

and

dehydrogenase

gave a single

of 33,503

other

(20)

dehydrogenase

fractions

(211, by polyacrylamide

centrifugation

on

cytosol

(Mr=37,000-40,003)

estradiol-178

placenta weight

liver

179-hydroxysteroid

specimens

discrepancies fact that

different

these

dehydroyenasc and

French

~13s ones,

ITable 1). The differsamples

populations

‘23;.

are obtained

REACTION MECHANISr~ OF THE ENZYME For

the oxido-reduction

between

androstenedione

and

testosterone, porcine testicular 17P-hydroxysteroid dehydrogenase preferred NADP~H~ to NAD(H). was used

When androstenedione

as a substrate, Km values for NADPH and

NADH were

determined to be 11 UM and 177

!_#I, respectively. Stoichio-

metric

consumption of NADPH exactly

analysis revealed that

corresponded

to the

androstenedione

amount

of testosterone

an equimolar

on

produced from

basis (24).

As far

as

we know, all of the reactions catalyzed by pyridine nucleotide-linked enzymes exhibit respect to the cofactors. NAD~P)H, one group

In the case of the oxidation of

of enzymes transfers only

prochiral center

hydrogen at the amide

absolute stereospecificity with

of the

the pro-R --

dihydronicotin-

to their substrate, while the other group

uses only

the pro-2 hydrogen. Information about the stereospecificity of

an enzyme

important

is

in the

elucidation

as of its molecular

of its

reaction mechanism

as well

at active-center.

To examine the stereospecific transfer

StTUCtUD?

of the hydrogen from NADPH to substrate by an enzyme, [4pro-R-3H]NADPH was -NAD?+

LlSiTlg

enzymatically

glucose-G-phosphate

3 HINADPH was other hand, [ii-pro-S--

using isocitrate dehydrogenase ; 25).

synthesized from dehydrogenase.

Qn

the

prepared from [4-?HlNADP' When 178-hydroxysteroid

dehydrogenase purified from porcine testes with androstenedione

[4-3H]-

in the presence

of

was incubated

these stereospe-

12

Jnano and Tamioki

cif'icallydifferent 3 H-labeled cofactors, only located at the 4--pro-S :_ _ position of the moiety

the tritium

dihydronicotinamide

of NADPH was transferred to testosterone (24). Lo-

cation of the

tritium in the testosterone molecule produced

was assigned

at the 17a-position, because the tritium

the testosterone remained

in its molecule after acetyla.-

tion, but was completely lost by chemical these

results, a transition

of

oxidation. From

state is proposed among andro-

stenedione, NADPH and testicular 173-hydroxysteroid dehydro~. genase, as shown in Figure 2. For oxidation 170

of estradiol-

to estrone, estradiol-170 dehydrogenase of human pla-

centa (26)

and chicken liver ;27) clearly transferred hydro-

gen to pro-S .-- - position of nicotinamide ring of the cofactor. In general, the hydrogen atom transferred to the a-face of the steroids molecule originates from pro-S side of the

Transition

state

Figure 2. Transition state for the reaction of androstenedione with NADPH catalyzed by 17fi-hgdroxysteroid dehydrogenase.

17/HWDRO,~STEROIDDEHYDROGENASE cofactors, occurs

whereas

transfer

from the pro-R

for

the

estradiol-178

of catalytic

(28). The dehydrogenase

amounts

system

between

the

the

and

because

and the cofactor

bound

hydrogenase covalently the

assay.

in the

However,

and

Also,

of

enzyme

presence and

On the other

testes was observed, to NADP+.

a

enzyme

of

trans-

containing

was obtained

bound

was devoid

no detectable dehydrogenase

either

that

by

[4-14Cl-

to

37Oc (30). the enzyme

of 17B-hydroxy-

to

transhydrogenation purified

from NADPH

One of the reasons

dehydrogenase

probably

reduction

using

(31) reported

from

to NAD+

NADP(H)

affinity relative

by

porcine

or from NADH

why the transhydrogenation

not occur would be due to the high steroid

During

activity.

hand,

17R-hydroxysteroid

the cyclic

3-(arylazido-!3.-

enzymic

modified

transhydrogenation

steroid dehydrogenase

the

with

[~-~IsP~-S-~H]NADH at

Villee

hydrogen

(29). When the estradiol-

covalently

of

of

of very high affinities

demonstrated

the

and NADP+

NADP(H)

[4-

Hagerman

that catalyzed

was

transfer

14C,17a-3Hlestradiol-178

bound

incubation

estrone

estrone

reaction

estradiol-170.

1713dehydrogenase was affinity-labeled 14 alanine)[ 4- Clestrone, the reversible covalently

steroid

was respon-

of estrogens

steroids

bound to the enzyme,

for estradiol-170

the

to NAD+ through

estrone

transhydrogenation,

remain

of

transhydro,genase

catalyzes

from the NADPH-generating interconversion

Y-face

dehydrogenase

steroid-stimulated

in the presence

the

the

side of the cofactors.

Human placental sible

to

13

did

of 178-hydroxyto

NAD(:I), as

14

Lnanoand Tamioki

suggested from the types of the

Km values.

cofactor are

Therefore, when these two

simultaneously present

in the

medium, it is speculated that NADP(H) would occupy the most accessible

cofactor-binding

sites

of

17P-hydroxysteroid

dehydrogenase, leaving almost no room for NAD(H) needed for the enzyme reaction related to the oxido-reduction. Furthermore, bioconversion of testosterone from androstenedione

by

the

17L3-hydroxysteroid dehydrogenase

presence

of

NADH was

the

the

decreased nearly proportionally by

increasing amount of NADP+ (24). of

in

initial velocities

By double reciprocal plots

for oxidation of testosterone

versus NADP+ concentrations in the presence of NADPH as the product

inhibitor,

the

17~-~ydroxysteroid

dehydroaenase

purified from porcine testes was competitively inhibited at both unsaturated and saturated concentrations of testosterone. Inhibition by NADPH was found to be a mixed type in the presence of an unsaturated amount of NADP' and variable amounts of testosterone, but was not observed in the saturated concentration of testosterone. Inhibition by the product steroid OP product pyridine nucleotide was competitive against

the

substrate

steroid OP

cofactor, respectively.

Also, no inhibition of the 17R-hydroxysteroid dehydrogenase activity by the product steroid and pyridine nucleotide was shown against the substrate pyridine nucleotide and steroid respectively at the saturated concentration of the cosubstrates. At strate,

the unsaturated

however,

concentration of

inhibitions by

the product

the

cosub-

steroid and

pyridine nucleotide were of mixed type against the substrate pyridine nucleotide and steroid, respectively (32). The characteristics of product inhibition suggest, as the reaction mechanism

of

the

dehydrogenase,

purified

one

of

testicular following

the

17P-hvdroxysteroid mechanisms:

rapid

equilibrium random Bi Bi system with two dead-end complexes, Theorell-Chance mechanism and ping-;oon$ mechanism. In the case

of the

complex

latter two mechanisms,

is to

no

ternary dead

end

be observed. The kinetic mechanism of the

testicular 17D-hydroxysteroid dehydrogenase is defined as a rapid

equilibrium

random

Bi 3i system with two dead end

complexes which are testosterone-enzyme-NADPH and androstenedione-enzyme-~ADP~ 'Figure 3). Formation of the ternary complex, testosterone-enzyme-NADPH, was observed

in fluo-

rescence experiments. Testosterone reduced the fluorescence increment of :JADPHdue to

of the 17p--hydroxg-

dehydrogenase.

steroid

NADP-E-T

the addition

_

NADPW-E-AD

ep

E-AD

_

11

Tl

II NADPH-E

E

E

NADPH-E-T

-

E-T

NADP-E-AD

?ZX$

NADP-E

TI -+

NADP-E-T

_

NADPH-E-AD

Figure 3. Equilibria for the bindin% of androstenedione;ADj, testosterone(T), NADPH and NADP to 173hgdroxysteroid dehydrogenase
16

Inano and Tamioki

Furthermore, the enzyme modified with 17B-bromoacetoxytestosterone was not able to increase fluorescence of NADPH to such an extent as the native enzyme. Those findings suggest the presence

of the

ternary complex

in which the

fluorescence of NADPH is intensified less than in the binary complex, quantitatively, the increase in emission of NADPH was depressed non-competitively by testosterone, suggesting the binding of testosterone to the binary complex of enzymeNADPH rather than the dissociation of NADPH from the enzymeNADPH complex due to the formation of an enzyme-testosterone complex. In

the human placental estradiol-17B dehydrogenase, the

reaction proceeded by an ordered Bi Bi mechanism, suggesting that the two eosubstrates were bound to the enzyme molecule in an obligatory order (29). For instance, steroids first bind to the enzyme and then pyridine nucleotide. The ternary complex,

estradiol-170-enzyme-NAD+,

forms transiently

and

then undergoes a hydrogen transfer to produce another ternary complex, estrone-enzyme-NADH. The reduced pyridine nucleotide is immediately released from the ternary Complex, and finally estrone dissociates from the binary complex. 3y the equilibrium isotope exchange procedure, Betz (33) concluded that the estradiol-170 dehydrogenase operated by random 9i Bi mechanism, suggesting that the order of binding is not predetermined. The activity of 178-hgdroxysteroid dehydrogenase bound to the microsomal

membrane

of

human and rat

testes :uas

17,bHYDROXYSTEROID

DEHYDROGENASE

17

stimulated markedly by product steroid. The optimal pH of 178-hydroxysteroid observed

as

5.8.

shifted to 7.4-7.8 reaction

mixture

17B-hydroxysteroid

dehydrogenase But,

the

for

optimal

androstenedione value

was

of the enzyme

in the presence of testosterone in the (34).

the

Also,

dehydrogenase

product

was

activation

demonstrated

of

by

the

increase of Vmax and decrease of Km for androstenedione. This would

be

consistent with

further

activation

of

an

active form of the enzyme rather than the conversion of its inactive form to an active state. Oshima -et -al

(35)

SW-

gested the presence of two binding sites in the molecule of the 17@-hydroxysteroid dehydrogenase of human testes, one specific

for androstenedione

and the

other specific for

testosterone, each serving as a binding site for the substrate and also as an activation site for the other active site. During the purification, 17L3-hydroxysteroiddehydrogenase of rat testes was separated from 17-ketosteroid reductase by differential (8).

fractionation with ammonium sulfate

Differences in the effect of sodium cholate on both

enzyme

activities

strongly

support

the

hypothesis

that

170-hydroxysteroid dehydrogenase and 17-ketosteroid reductase are two distinct enzymes. However, the reduction of androstenedione in the presence of NADPH proceeded at the same rate as the oxidation of testosterone in the presence of NADP+ by the 170-hydroxysteroid dehydrogenase purified from porcine testes (24).

18

Inane and Tamioki

STRUCTURE No region

information was

about

obtained

from

because

hydrogenase, tion,

OF ACTIVE-SITE the

OF THE ENZYME structure

testicular

instability

of the purified

estradiol-17D

dehydrogenase

placenta

by

simpler

procedures

However,

the size of the crystal

nase was insufficient Since

1973,

estradiol-17L3 modification

the

(8,9). been

(36)

topography

affinity

has

catalytic

of purifica(b), and the

purified

and

from

crystallized

of estradiol-178

of

the

been

labeling

de-

On the other hand,

for X-ray diffraction

dehydrogenase and

method

in the tissues

enzyme has

the

17!3-hydroxysteroid

of the complicated

its very low concentration

in

human (37).

dehydroge-

analysis.

active-site studied

in the

by

chemical

(Figure 4).

Affinity

Figure 4. The proposed orientation of estrone and cofactor within the active-site of estradiol-17B dehydrogenase.

17~HYDROXYSTEROID

labeling

of

the

indicated

progesterone to

inactivated

the

active-site reported

that

with

being

three

to

binding

the

participated

D-ring

second third

step

residues

active-site

residue

one

with

and

those methyl

estrone-3-

was

located present

Lysine residue

located

bound

was

an

itself

the

regions

other

the

underwent

the

residues

or

catalytic

of

the

by

ether,

near

One

region,

the

site, and (45,46).

of the A-ring

but

labeling was

the

(44).

active-site

affinity

near

(42,43).

transfer at

as

of

cofactor-binding

not

of the with

4-

essential

(47).

a-dicarbonyl

steroid,

dehydrogenase

located

identified

dehydrogenase

the

methyl

estradiol-17B residues

two

identified

at

detected

16-Oxoestrone,

D-ring

in the vicinity

for the enzyme activity

two arginine

also

in

were

hydrogen

outside

bromoacetamidoestrone

inactivate

the

located

was

steroid

in

dehydrogenase

near the A-ring

(411, and

were

was

which and

estradiol-17B

of

one

A-ring

residue

reversible

cysteine

in the

2-bromoacetamidoestrone

residues

the

One histidine

Cysteine

identical

bound

carboxyl-

residues

site of estradiol-178

histidine

proximate

steroid.

ligand

lysine

are

were

(40).

The steroid-binding contains

The

lla-bromoacetoxyprogesterone

(391,

bromoacetate

dimer. and

19

6B-bromoacetoxy-

of ligand

results

for alkylation

with

2 moles

histidine

The

(38).

enzyme

enzyme

cysteine,

methylated

ether

placental

DEHYDROGENASE

around

by

was

able

modification

the active-site

to of

of the

20

Inanoand Tamioki

enzyme (48). The functional arginine residues of the enzyme wepe involved in binding to the 5'-diphosphate moiety and s

2'-phosphate moiety of cofactors. It is generally accepted that

the arginine residues

in

the catalytic region

of an

enzyme can serve as a positively charged site for recognition of negatively charged substrates and the anionic moiety of the cofactors. Since estradiol-17P dehydrogenase was l-ethyl-3-(3-d~methylaminopropyl~carbodi~mide

inactivated by (EDC) in the

buffer with high ionic strength, carboxyl groups or the side chain of aspartic (or glutamic) acid residues were an essential for the conversion of estradiol to estrone and were partially buried in the enzyme molecule (49). The enzyme activity was rapidly decreased by EDC in the presence of the oxidized cofactors, NAD(P)', but not In the presence of the reduced

forms. These

findings

suggest that

the

positive

charge of nicotinamide Nl of the oxidized nucleotides plays an important role in the increment of the inactivation by EDC. Tetranitromethane inactivated the native estradiol-I'7R dehydrogenase more rapidly than sulfhydryl-masked enzyme obtained from the treatment with 5,5'-dlthlobis(2-nitrobenzoic acid).

Both

chemical

modifications

followed pseudo-first

order kinetics, because tetranitromethane modified tyrosine residues at or near the active-site of estradiol-17B dehydrogenase. The nitration of tyrosine residue was complete-

17&HYDROXYSTEROIDDEHYDROGEivASE

21

His-Phe-Thr-His-Ile-Asp-Thr-Arg-Asp-Leu-NHCO-R’ Figure 5. Amino acid sequence of catalytic region in estradiol-17B dchydrogenase. iy blocked by NADPCH) and partially by NADCH), suggesting that the 2'-phosphate moiety of NADP(H) disturbs the chemical modification of tyrosine residues probably located at the cofactor-binding site (50,51). Furthermore, estradiol-

1713 dehgdrogenase was affinity-labeled at its substratebinding site by 3-(arylazido-a--alaninelestrone (30), and at its

cofactor-binding

site

by

5'-p--fluorosulfonylbenzoyl

adenosine (52). Burdock -et al

C41) have schematically represented the

sequence of amino acid residues composing the active-site of estradiol-17D dehydrogenase (Figure 5). A computer graphic model of the active-site of the estradiol-173 dehydrogenase suggested

that

the

enzyme

binds

a

progestin

substrate

inverted 1.80'relative to the estrogen to accommodate stereospecific

oxido-reduction at the

respectively (38). Why do

17B- and

20a-positions,

estradiol-17B dehydrogenase and

ZOa-hydroxysteroid dehydrogenase share a common locus on a single protein ?

How are the two enzyme activities requlat-

ed ? To solve these problems will require knowledge of' the shape and flexibility of the active-site and how the steroid molecules fit into it. A likely approach may well. require some combination of the foIlowing techniques: X-ray crystal-

22

Inanoand Tamioki

lography

will

hydrogenase flexes

to

help

and

explain

how

permit

the

both

Computer

active-site.

the

tertiary

steroid

hormones

use

of

will

active-site

help

hormone

into

us

the

determine

of the

active

sites

amino

the

region

de-

enzyme same

of

the

of the approach

Finally,

to more

as it binds to the active

of

to fit in the

an estimate

which

estradiol-17B

structure

active-site.

labeling

of

molecules

modeling

amino acid chain would permit the

shape

on

acids

expanding the are

of the

hormone near

the

cleft of the enzyme.

ACKNOWLEDGMENTS We are indebted to Dr. H. Ishii-Ohba, Dr. K. Hamana, Dr. H. Idakagawa, Dr. H. Fujita and Mr. K. Kawakura for their valuable advice and cooperation and to Ns. J. HayashiyamaOdaka for her technical assistance. Supports by Grant-in-Aid from the Ministry of Education, Science and Culture of Japan and by a promoted research grant of Nation?1 Institute of Radiological Sciences are acknowledged.

NOTES AND REFEREUCES is paper 3 of the series This Hydroxysteroid Dehydrogenase." To whom correspondence

'Porcine Testicular Paper 7 is ref.32.

171%

should be addressed.

Ferguson, 14. 14. and Baillie, A. H., Hart, '4. D. 1 The testis and epididymis, in: Developments in ----Steroid __ :iis_--tochemistry, Academic Press-,-Londo-n,-~1~66) pp 5'1-55. Kurosumi, ?I., Ishimura, K., Yoshinaga, Y., Fujita, 1~. and Tamaoki, B. : Immunocytochemical localization of 17D-hydroxysteroid dehydrogenase in porcine testis. HISTOCHEWISTRY 82, 287-289 (1986). :'lurono,E. P. and Payne, A, H.: Distinct testicular 17ketosteroid reductase, One in interstitial tissue and one in seminiferous tubules, Differential modulation by testosterone and metabolites of testosterone. 3IOCHIy.4. BIOPHYS. ACTA 459, 89-100 '1976). Inane, H., Inano, A. and Tamaoki, B.: Studies of enzyme reactions related to steroid biosynthesis II. S.ubmicroSOrlSl distribution of the enzymes related to androgen

23

5.

6.

7.

8.

production from pregnenolone and of the cytochrome P1-) 450 in testicular gland of rat. J. STEROID BIOCHE?/I. 83-91 :19701. Xachino, A., Nakano,H. and Tamaoki, B. : Influence Of physical and chemical treatments upon the microsomal enzymes of testes related to androgen biosynthesis. ENDOCRINOL. JAPOIJ.&6_, 37-46 (1969). Tamaoki, B. : Purification and properties Inano, I$. and of NADP -dependent 170-hydroxysteroid dehydrogenase solubilized from porcine testicular microsomal fraction. EUR. J. BIOCiiEi?. g, 13-23 (1974). Inano, H., Ohba, H. and Tamaoki, 5.: Porcine testicular 174-hydroxysteroid dehydrogenase: Affinity chromatography with dye-1i;and asarose and demonstration of multiple forms of the enzyme. J. STEROID BIOCHEr4. 12, 13471355

11931;.

Bogovich, K. and Payne, A. H.: Purification of rat testicular 17-ketosteroid reductase, Evidence that 17-ketosteroid reductase and 17!3-hydroxysteroid dehydrogenase are distinct enzymes. J. BIOL. CHEM. 255, 5552-5559 (1930).

9.

Inano, H., Hayashiyama, J. and Tamaoki, B.: Solubilization of A'-38-hydroxysteroid dehydrogenase with As-A'isomerase and 178-hydroxysteroid dehydrogenase from rat testicular microsomal fraction of several detergents. J. STEROID SIOCKEX. 16, 537-593 (1952). 10. Wan, EC. J. and EnSel, L. L. : The interconversion OQ estrone and estradiol by human tissue slices. ENDOCRIXOLOGY 52, 237-291 (1953;. Il. Karavolas, H. J., Baedecker, X. L. and Engel, L. L.: Human placental 17D-estradiol dehydrogenase, V. Purification and partial characterization of the diphosphopyridine nucleotideitriphosphopyridine nucleotide)-linked enzyme. J. BIOL. CHEM. 245, 4948-4952 (1970). 12. Strickler, R. C. and Tobis, B.: Estradiol 173-dehydrogenase and 20a-hydroxysteroid dehydrogenase from human placental cytosol : One enzyme with two activities ? STEROIDS 36, 243-253 :1930). :Jarren,J. C.1 Purification and 13. Henderson,-L. L. and characterization of epimeric eatradiol dehydrogenase(l7a and 17D; from equine placenta. BIOCHEI4ISTRY _-2 23 486-491 lir.

15.

16.

i(l984). i,lichel,

F. , i'icolas,J.-C. and Crastes de Paulet, A.: 17-8-Aydroxysteroid dehydrozenase of the sheep ovary: Purification, properties and substrate binding site. BIG'XI14IE 57, 1131-1140 (1975). Watson, D.-g., Harvey, X. J.+and Dean, P. 3. C.1 The selective retardation of XADP -dependent dehydrosenase by immobilized Procion Red HE-3B. BIOCHEI4.J. 173, 591-596 il9731). Antoun, C. R., Brslez, I. and Williamson, D. G.1 A 17Bhgdroxysteroid dehydrogenase of female rabbit liver cyJ. Z>, toso1. BICC;IE:II. 333-330 (1985).

24

Inane and Tamioki

dehy17. Heyns, W. and DeMoor, P.: A 3(17)D-hydroxysteroid drogenase in rat erythrocytes, Conversion of 5a-dihydrotestosterone into 5a-androstane-3S,17B-diol and purifienzyme by affinity chromatography. BIOcation of the CHIM. BIOPHYS. ACTA 358, l-13 (1974). 18. Inano, H., Tamaoki, B., Hamana, K. and Nakagawa, H.: Amino composition and immunochemical properties acid of porcine testicular 178-hydroxysteroid dehydrogenase. J. STEROID BIOCHEM. u, 287-295 (1980). form of 19. Inano, H., Kawakura, K. and Tamaoki, B.: Active 17B-hydroxysteroid dehydrogenase of porcine testes. .T. STEROID BIOCHEM. 8, 787-789 (1977). 20. Liu, D. K. and Kochakian, C. D.: Heterogeneity of guinea pig kidney 170-hydroxy-C -steroid dehydrogenase activig&P electrophoresis. STEROIDS 2, ty observed by disc 721-729 (1972). 21. Pollow, K., Lubbert, H. and Pollow, B.: Partial purification and evidence for heterogeneity of the cytoplasmic 17B-hydroxysteroid dehydrogenase (173-HSD) from normal human endometrium and endometrium carcinoma. J. STEROID BIOCHEM. 1, 315-320 (1976). 22. Burns, D. J. W., Engel, L. L. and Bethune, J. L.: The subunit structure of human placental 17!3-estradiol dehydrogenase. BIOCHEM. BIOPHYS. RES. COMMUN. 'l'i,786-792

(1971,. Groman, E. V.1 Human placental 17R23. Engel, L. L. and estradiol dehydrogenase: Characterization and structural studies. RECENT PROG. HORM. RES. 30, 139-169 (1974j. 24. Inano, H. and Tamaoki, 5.1 Relationship between steroids and pyridine nucleotides in the oxido-reduction catalyz17B-hydroxysteroid dehydrogenase purified ed by the from the porcine testicular microsomal fraction. ESIR. .T. BIOCHEM. 53, 319-326 (1975:. of hydrogen transfer 25. Abul-Hajj, Y. J .: Stereospecificity by steroid A"-5a- and A"-5C-reductases. STEfrom NADPH ROIDS 20, 215-222 (19723. of hydro26. Jarabak, ,J. and Talalay, P.: Stereospecificity by pyridine nucleotide-linked hydroxytransfer gen J. BIOL. CHEM. 2147-2154 dehydrogenase. steroid 235,

(1960). 27. George, J. M., i)rr, J. C., Renwick, A. G. C., Carter, ?. of hydroi;en transand Engel, L+ L.: The stereochemistry NADP by enzymes acting upon stereoisomeric subfer to strates. BIOORG. CHEM. 2_, 140-144 '19733. 28. Talalay, P., Hurlock, B. and Itiiiliams-Ashman, H. 1;.1 i-1n a noenzymatic function of estradiol 17R-dehydro.renase. PROC. NATL. ACAD. SCI. U.S.A. 4'& 862-384 '19591. 29. Warren, J. C. and Crist, R. D.. Site specificity and mechanism of human placental 17B-hydroxysteroid dehydrogenase. ARCH. BIOCHEM. BIOPHYS. m, 577-584 (19673. labelin? of 30. Inano, H. and Engel, L. L.: ?hotoaffinity human placental estradiol dehydrogenase with 3-:arylazido-B-alaninejestrone. .J. BIl?L. CHEM. '55, 7694-7599

17~HYDROXYSTEROIDDEHYDROGENASE

25

,193oj.

D. D. and Villee, estrogen-sensitive placental dehydrogenase. estradiol-170

31. Hagerman,

C. A .: Separation of human from transhydrogenase J. BIOL. CHEM. --234, 2031-

2036 (1959). 32. Ohba, H., Inano, H. and 33. 34.

35.

36.

Tamaoki, B.: Kinetic mechanism testicular 17!3-hydroxysteroid dehydrogenase. of porcine J. STEROID BIOCHEX. 17, 381-336 (1982). Reaction mechanism of 17B-estradiol dehydroBetz, G.: genase determined by equilibrium rate exchange. J. BIOL. CHEM. 246, 2063-2068 (19713. Oshima, H. and Ochiai, K .: 3n testicular 17B-hydroxysteroid oxidoreductase, Product activation of testosterone formation from androstenedione in vitro. BIOCHIIJ. ----BIOPHYS. ACTA 306 _, 227-236 (1973). Oshima, H., Yoshida, K. and Troen, P.: A further study of 17D-hydroxysteroid oxidoreductase in the human lilechanism of -in vitro activation. ENDOCRINOL. testis: --JAPON. 27, 107-115 (1980). I.lendoza-Hernandez, G., Rendon, J. L. and Diaz-Zagoya, J. c.: A single procedure for purification of estradiol 17B-dehydrozenase. BIOCHEX. BIOPHYS. RES. corilrilu~.126, 477-_-

431 (19853. 37. Chin, C. C., Dence, J. B. and 38.

39.

40.

41.

42.

43.

Warren, J. C .: Crystallization of human placental estradiol 17!3-dehydrogenase. J. BIOL. CHEX. 251, 3700-3705 ;lg76). -Thomas, J. L. and Strickler, R. C .: Human placental 17Destradiol dehydrogenase and 20a-hydroxysteroid dehydroStudies genase, with 6R-bromoacetoxyprogesterone. J. BIOL. CHE!?. 253, 1537-1590 (1983). Chin, C. C., Asmar, P. and Warren, J. C.: Synthesis of 2-bromoacetamidoestrone methyl ether study of the and steroid-binding site of human placental estradiol-175 dehydro%enase. J. BIOL. CHEX. 255, 3660-3664 (1980). Thomas, J. L., Asibey-Berko, E. and Strickler, R. C.: The affinity alkglators, lla-bromoacetoxyprogesterone and estrone-3-bromoacetate, modify a common histidyl residue in the active site of human placental 17B,20ahgdroxysteraid de!lydrogenase. J. STEROID BIOCHEFI. 25 __) 103-193, ;1936j. :durdock, (G. L., Chin, C. C., Offord, R. E., Bradshaw, R. A. and IJarren, J.C.: Buman placental estradiol 17B-dehyof a single histidine residue drogenase, Identification affinity-labeled by both 3-bromoacetoxyestrone and 125bromoacetoxy-4-estrene-3,17-dione. J. BIOL. CHEM. 258 ___) 11460-11464 (1933.1. :,lurdock,'3. L., Chin, C. C. and 'Warren, J. C.: Human placental estrsdiol 1784ehydrogensse: Sequence of histidine bearin; pegtide in the catalytic region. BIOCi15::/IISTR71 25, 641-biiij(1936). Chin, C. C.,Murdock, G. L. and Warren, J. C. 1 Identification of two histidyl residues in the active site placental estradiol Of human 17!3-dehydrogenase. BIO-

28

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

Inane and Tamioki

CHEXISTRY 2l_, 3322-3326 (1382). Biellmann, J.-F., Branlant, G., :jicolas, J. C., Pans, M Descomps, B. and Crastes de Paulet, A. 1 Alkylation of'estradiol 17a-dehydrogenase from human placenta with 3-chloroacetylpyridine adenine dinucleotide. EUR. J. BIOCHEM. 63, 477-481 :1976). Pons, ?I.,-Nicolas, J. C., Boussioux, A-M,, Descomps, B. of the esand Crastes de Paulet, A.: Affinity labelin. with subtradiol-17D dehydroq?nase from human placenta strate analogs. EUR. J. BIOCHEX. 68, 335-394 (1976). Thomas, J. L., LaRochelle, X. C.,-Asibey-Berko, E. and Strickler, R. C.: Reactivation of human olacental 178and 20a-hydroxysteroid dehydro.