Soybean lipoxygenase-catalyzed oxidations by linoleic acid hydroperoxide: Different reducing substrates and dehydrogenation of phenidone and BW 755C

Vol. 151, No. 1, 1988 February 29, 1988

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 339-346

SOYBEANLIPOXYGENASE-CATALYZED OXIDATIONSBY LINOLEIC ACID EYDROPEROXIDE : DIFFERENTREDUCINGSUBSTRATES ANDDEHYDROGENATION OF PHENIDONE AND BW755C D. MANSUY, C. CUCUROU, B. BIATRY and J.P.

BATTIONI

Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques (UA 400 CNRS), Universite Ren6 Descartes, 45 Rue des Saints-Peres, 75270 PARIS CEDEX 06, France Received

January

15,

1988

Summary : Phenidone is not a substrate for dioxygenation by soybean lipoxygenase-1 (Ll) but reduces LlFe(II1) into L Fe(II), as shown by EPR spectroscopy. Ll catalyzes the oxidation of phen 1 done by 13-HPOD, the hydroperoxide formed by dioxygenation of linoleic acid by Ll, with formation of 4,5-dehydrophenidone. Two moles of 13-HPOD are used per mole of phenidone dehydrogenated. Other pyrazoline derivatives such as BW 755C, but also, in a more general manner, different compounds containing phenol, aniline, hydrazine, hydroxylamine or hydrazide functions act as reducing substrates for decomposition of 13-HPOD by Ll. @ 1988 Academic Press, Inc.

Interest years

in

the mechanism

by the discovery

of leukotrienes

which

hypersensitivity

(1).

for

detailed

amounts

of the have

RH = linoleic

are

(2).

since

available It

of lipoxygenases rale

catalyzes

by Ll unit

are

(scheme (2)

1).

site

agents,

analogs of our

and undergo

we show herein

phenidone

and BW 755C,

cyclooxygenases only

to act

catalytic

dioxygenation

phenidone

(5,6),

fatty

(3),

current

studies (i)

Ll

two classical into

the

and BW 755C but

acids

the

possessing

corresponding also

large

scheme acid

1 with

to

very

few

dioxygenation

cycle

a cis,cis-1,4-pentadiene (4).

oxidation inhibitors

(see

So far,

complete

on compounds catalyzes

cycle

in

of linoleic

and phenylhydrazones

a Ll-catalyzed that

for

biosynthesis interesting

purified

(13-HPOD).

last

and immediate

particularly

on its acid

all

is

can be easily

as substrates

They are

or their

As a part active

known

(Ll)

these

in the

in inflammation

it

13(S)-hydroperoxy-9Z,llE-octadecadienoic compounds

has been stimulated

lipoxygenase-1

studies

data

acid)

implication

an important

Soybean

mechanistic

and clear

of lipoxygenases

that

can interact

by 02 or other the

oxidation

dehydrocompounds, containing

Ll

by 13-HPOD of

of lipoxygenases

many compounds

with

oxidizing and and (ii) phenol,

not aniline,

ABBREVIATIONS : L

: soybean lipoxygenase-1, 13-HPOD : 13(S)-hydroperoxySZ,llE-octadecadi&noic acid, DETAPAC : diethylenetriamine-pentaacetic BW 755C : 3-amino-l-[3-(trifluoromethyl)phenyl]-2-pyrazoline, NDGA : nordihydroguaiaretic acid, phenidone : I-phenyl-3-pyrazolidone.

339

acid,

0006-291X88 $1.50 Copyright 0 1988 by Academic Press. Inc. All rights of reproduction in any form reserved.

Vol. 151, No. 1, 1988

Scheme

hydroxylamine

are

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1 . Catalytic cycle of dioxygenation by Ll(RH = linoleic acid, steps 1, 2, 3) and catalytic cycle of oxidations by L and 13-HPOD (= LOOH) (RH = reducing agent as phenidone, s 1 eps 1 and

hydrazine, and

BIOCHEMICAL

oxidized

by

or 13-HPOD

hydrazide in

functions

the

presence

reduce of

LlFe(III)

into

1’).

LlFe(II)

Ll.

MTRRIALSANDlWl'RODS BW 755C was a gift from Wellcome Lab. and ketoprofen from Rhane-Poulenc. 1-phenyl-3-hydroxy-pyrazole (4,+dehydrophenidone), 1-phenyl-3-amino2-pyrazoline and 1-phenyl-2-methyl-3-pyrazolidone were respectively prepared according to ref. 7, 8 and 9. N-hydroxyamphetamine, 4lnethylbenzaldehyde-4’-bromophenylhydrazone and N’-phenylbenzoylhydrazide were prepared by previously described procedures (10,11,12). All other chemicals were purchased from commercial sources at the highest level of purity available. Linoleic acid (99 %, from Sigma) was purified-ynder argon as cm ) was reported previously (4). 13-HPOD (Amax = 234 nm, E = 25 mM prepared enzymatically by a described procedure (13) and stored under argon in isopropanol at -18’C. L1 was purified from seeds (KiTg S”y variety) by a described method (14,4) (Xmaxl= 289 nm, E = 160 mM cm ). Its specific activity was about 120 pmol.min .mg . W-visible measurements were done with an Uvikon 810 Kontron spectrophotometer. Reactions between L 13-HPOD and various compounds were performed at 20°C in 50 mll tris-aceta H9 containing 0.1 mM DETAPAC in 3 ml i’ e buffe -5 p M solutions in methanol and we cuvettes. Compounds were added from 10 verified that equal amounts of pure methanol had no significant effects on L . Oxygen uptake was measured with a Gilson oxygraph equipped with a Clark’ electrode in a 1.2 ml thermostated (19°C) cell under conditions identical to those used for spectrophotometric assays. RPLC aoalysis (after 10 min) of anaerobic incubations of 10 PM phenidone with 10 PM 13-HP5 and 0.5 PM L in 3 ml buffer pH9 were done after acidification to pH5 by addition of 1 A citric acid to stop phenidone oxidation. Separations were made on a Nucleosil Cl8 reverse-phase column (Bischoff) For phenidone and its dehydroderivative, an isocratic elution (5 pm, 12.5 cm). mobile of a 35:65 mixture of CH CN : 0.1 M ammonium acetate buffer pH4.6 (1 ml per min) and UV detection at 25 2 nm were used. For 13-HPOD, elution mobile was a 70: 30 CH3CN : same buffer mixture and UV detection at 234 nm.

RESULTS 100 dioxygen (15).

/&M solutions at

In

the

a very presence

of low

phenidone rate

of

Ll,

in

because even

50 q M tris-acetate of

in

the large

340

slow amounts

buffer

autoxidation (0.2

PM),

pH9

consume

of

this

no

increase

compound of

O2

BIOCHEMICAL

Vol. 151, No. 1, 1988

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

96.1

5bo

10’00

MAGNETIC

Figure

1 . Effect

of

94.3

phenidone

l&o

FIELD

on the

2cioo (CNJSS)

EPR spectrum

of

ferric

Ll

:

A : ferric enzyme (0.5 mM1 obtained by addition of 1 mole of 13-HPOD per mole treatment with 1 molar equivalent of phenidone, C : of L Fe(II1, B : further then: further-treatment with 5 molar equivalents of 13-HPOD. Experiments were done under anaerobic conditions in 50 mM tris-acetate pH9 buffer, 0.1 mM DETAPAC. Spectra were recorded on a Brucker ER 220 D spectrometer at 9.34 GHz and 10 K, using an Oxford Instrument continuous flow cryostat, a Hall probe and a Hewlett-Packard frequency meter (100 KHz modulation frequency).

consumption

occurs.

This

dioxygenase

activity

of

the

catalytically

complete which 13-HPOD

(Z),

few

of

two

LlFe(III) of

state.

These

phenidone

by

steps,

of

phenidone).

Accordingly,

of

Ll

as

judged

to

according

LlFe(II)

by

the

13-HPOD

fast

is that

ferrous

EPR

characteristic

phenidone as

mixture

of (steps

reacts

1 and

of

341

1’ with

its

This

addition

of

by

LlFe(II1) scheme

1 with

phenidone

in

at

and

234

of into

the RH = the

nm upon

a

Cl

oxidation

consisting

of

UV peak

of the

an

cycle

the

of

regenerates Ll

by

LlFe(III),

1).

a further

by

phenidone

shown

of

the

readily

equivalent (Fig.

catalytic

rapidly

disappearance

one

fully

catalysis

a shortened

was

signal

of

for

reduces This

by

reversible

reaction

a substrate

state.

LlFe(II)

equivalent is

not phenidone

its

a possible to

13-HPOD

found

resting

oxidation by

the

one

suggest

one-electron

reoxidation

of

LlFe(I1)

13-HPOD data

to

g = 6.1

of into

13-HPOD

the

the

phenidone

we

Ll

oxidation

addition

equivalents

ferric

ferric

by

upon

that

However,

of

obtained

reduction

Ll.

active

disappearance was

indicates

presence

Vol. 151, No. 1, 1988

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A290

0.15

0.1 0

0.05

,_ ,c,

B

-

0

2 . Time

of

Ll-catalyzed

oxidation

of

phenidone

2

(min)

TIME

courses

I 15

‘ 10

5

(min)

TIME

Figure

+13-HPOD

by

13-HPOD.

A : disappearance of 13-HPOD during 3 successive additions of 8 /.&M 13-HPOD to < solution containing initially 10.3 PM phenidone and 0-f pI$L1. 1 : appearance of 4,5-dehydrophenidone (at 290 nm, E= 18 mM cm ) upon 3 successive additions of 13-HPOD (6.9 PM each) to a solution containing 9.3 PM phenidone and 0.2 /JM L . Additions of Ll together with 13-HPOD were necessary because of L1 inactiva 1.Ion during the reaction.

reaction

of 10/.4M 13-HPOD with

1).

Under

the

rate

identical

conditions

of decomposition

of disappearance

of

L1 and of phenidone

Table

10 /.4M phenidone but

of 13-HPOD is

13-HPOD is (only

1 . Dependence

of

directly

for

I3-m0D

WH)

wf)

0 0.2 0.2 0.2 0.2 0.3 0.4 0.2 0.4

10 10 10 10 10 10 IO 30 30

Conditions e234

aerobic

=25

in r&l-'

Materials CIII-~.

measurements

absence

initial L1

rate and

of either

negligible

of 13-HPDD phenidooe.

Phenidone (lul)

10 0 3 5 10 10 10 100 100

10 /.&M for

2 and table

or phenidone, The initial

rate

concentrations

of

the

latter

consumption

oo

concentrations

Initial

rates

QOI/min)

aerobic


anaerobic conditions I 1

0 co.

I .4 2.5 4.0 6.0 8.0 13 26

and Methods. Rates are determined Results are averagesof four independent and two independent experiments for

342

1).

to the

below

(Fig. Ll

(table

proportional

concentrations

of

L-l

in the

and 0.2 FM Ll

1

4.2

13 27

at

234 nm using experiments for anaerobic measurements.

BIOCHEMICAL

Vol. 151, No. 1, 1988

compound).

This

anaerobic

rate

is

conditions

almost

(table

HPLC analysis of phenidone

with

shown).

This

compound

a 75-80

% yield

under

that

anaerobic This

of

in good

agreement

of phenidone with of

of

also,

these

the

are

by

I3-EPOD

which

when

appeared

phenols

such

as guaiacol

all

compounds

the

hydrazone,

highest

as the

rates

hydrazide

benoxaprofen Ll-catalyzed consumed

L1

is

most

reactive

several

in the

appeared oxidation

the

of

2).

preparation

as inactive

in

by 13-HPOD,

of L1 and

per

if

this it

min). 343

are

This

in

active

also and give them

assay. were

is

found

to

the

case

for

agreement

with

a

of this

also

this

series

in

compounds

manuscript.

this

series

with

NDGA and but

lower

simple

rates.

From

and N-hydroxyamphetamine containing

function It

naproxen,

As

contrary,

in

various

cycle

itself.

On the

inactive

for

catalytic

omitted.

compounds

like

substrates

For

function,

of Ll.

as

CF3 group (data

(table

(16)

act

shown)

E are

presence

of

respectively),

of phenidone

so far , para-aminophenol but

2.05

not

present

the

13-HPOD to

of phenidone.

are

during

of

to those

or a diphenol

of cyclooxygenase,

per pmol

vas

of

a stoichiometry

and

to

without

compounds

or hydroxylamine

13-HPOD consumption inhibitors

many

and vitamin

tested

(2.1

to that

similar Ll

oxidation

gave

4,5-dehydroderivatives if

the

of

to the shortened

analogue

very

13-HPOD

of phenidone,

additions

phenidone

was compared

a phenol

appear

gave

1’)

manner,

pyrocatechol the

it

by HPLC. of

and 4,5-dehydrophenidone

containing

(17)

of phenidone

by

either

L1-catalyzed

progressive

obtained

negligible

In a more general

report

with

of phenidone

phenidone

appearance

13-HPOD according

by

corresponding

rates

oxidized

formed

mole

excess

Both methods

derivatives

1 and

N-methylphenidone

be

per

of

for

or the 2).

mole

result

13-HPOD consumption

compounds

not

HPLC experiments,

spectroscopy,

previously

2, BW 755C and its

to the

rates

phenidone,

and is

to an initial

(16),

(Fig. per

various

1 (steps

oxidation

= 290 nm) upon

13-HPOD consumed

in table

oxidized

under

(data

reaction

consumed

in UV-visible

described

oxidations

shown

between

From these

progressively

(Amax

ability

of scheme

of the

L1-catalyzed

by 13-HPOD

excess

2 moles

the

13-HPOD added

4,5-dehydrophenidone

Ll-catalyzed

performed

of 4,5-dehydrophenidone

of 13-HPOD are

by following,

to a technique

The

reactions

phenidone.

moles

of

N-alkylhydroxylamines an initial

is

conditions.

of

according

reaction

13-HPOD and show the disappearance

product

on starting

2 + 0.2

determined

consumption

from of

formation

the major

stoichiometry

was also

coming

consumption

concomitant is

when the

1).

the

based

was estimated

identical

of incubates

13-HPOD and LL confirm

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

is

an hydrazine, were

noteworthy

indomethacin, assay

exists,

(i.e. is

found that

to induce well-known

ketoprofen their

below

rate

of

1 pmol

of

and 13-HPOD

Vol. 151, No. 1, 1988

Table

2

BIOCHEMICAL

.

LI-catalyzed

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

reduction

of

134iPOD

Initial rate a (ti -9

Substrate

compounds.

Initial

Phenylhydrazine 2-hydrazinopyridine

10

52

N-hydroxyamphetamine

clb

170

Cl

(Vitamin

4-methylbenzaldehyde-4’bromophenylhydrazone

<5 13 90 (5 140 13 Cl 170 Cl

Gua'iacol

Pyrocatechol Resorcinol NDGA DL-CY-tocopherol Aniline 4-aminophenol 4-acetamidophenol

various

Substrate

68 68 68

Phenidone BW 755C 1-phenyl-3-amino-Z-pyrazoline I-phenyl-Z-methyl-3-pyrazolidone I-phenyl-3-hydroxy-pyrazole (4,5-dehydrophenidone) Phenol

by

E)

(acetaminophen)

43

8

N’-phenylbenzoylhydrazide Naproxen Indomethacin Ketoprofen Benoxaprofen

Cl Cl Cl
a)Initial rates of 13-HPOD disappearance (decrease of the 234 nm band), with 0.2@ are expressed inpmol per min and perpmol Ll. 30 PM 13-HPOD and 100 PM compound, Consumption of 13-HPOD under the indicated conditions with compounds that gave significantly different from the rate of rates ~1 was verified by HPLC. b)Not I3-HPOD consumption with L1 alone under identical conditions (22).

Ll,

DISCUSSION

Phenidone However, and

and

they

1’

of

are

1)

dehydrophenidone cycles

mole

of

and

for are

consistent

formation

may

scheme

1)

into

its

with

(=

13-HPOD)

for

fate

of

is

LO’

the

explains

(Fig.

2A).

the

two

moles

required

of

LlFe(I1)

with

which

has of

1 correspond

the to

the 234

the

(and

one

mole of

closest

conjugated

nm band

Ll

(steps

LO’

1

the of

so

two

short

oxidation

stoichiometry

formation on

lost

of

Ll.

of

two-electron The

need

by

mechanism

13-HPOD the

of

formation

detailed

for

reported

cycle

with

dehydroderivative.

disappearance

Equation

Whatever

addition

radical the

13-HPOD

be,

dioxygenation

catalytic

by

are

intramolecular

epoxyallylic

This

of

for

a shortened

previously

oxidation an

substrates

oxidized

of

phenidone

is

an

not

dehydroderivatives.

catalytic

to

are

substrates

scheme

corresponding

one

BW 755C

of

of

of

eq.

LOOH

(2,18).

The

major

double

bond

leading

diene

system

(2).

observed

in

a “hydroperoxidase-like”

1

such

reactions

activity H I

L1

of

Ll

in

the

activity

in

the reaction. of

sense

that However,

horseradish

an

hydroperoxide it

is

peroxidase

CH+ 2 LO'

acts

different

from

or

prostaplandin

344

as the

a cosubstrate classical H synthase

(eq.

and

is

1)

reduced

hydroperoxidase for

which

the

BIOCHEMICAL

Vol. 151, No. 1, 1988

hydroperoxide

is

oxidizing

reduced

equivalents

peroxidases

for

catalyze

all

substrates

for

linoleic

itself

similarly.

case

many

and

the

a substrate

to

provides

its

instance, and

alcohol

of

structural

possible

two

these

BW 7556

derived

two

by

from

the

a good

substrate

is

Ll

case

13-HPOD

(2)

and

results

not

only

the

of of show

or

our

of

these to results necessary

H synthase.

the

also

a compound

factors

prostaglandin latter

that

behave

preliminary

structural

for

2)

products for

from

be

but

(table

important

activity

of

to

the

Ll

for

dioxygenation

able

enter

cooxidations

of

compounds

the

lipoxygenase-Fe(II1)

precursors

inactive

of

More the

ACKNOWLEDGMENTS studies

and work

would is

: We thank

I.

is

(21)

L1

interesting active

in

the

for

For whereas

act

as

by

tend

it

know

or

because

of

linoleic

acid

and

reduce the

was

this

efficiently enzyme

recently

in

3. 4.

5. 6. 7.

shown

phenomenon

is

a grant

France) for

one

for for

us

her (C-C).

Samuelsson, B. (1983) Science 220, 568-575. Vliegenthart, J.F.G., and Veldink, G.A. (1982) in “Free Radicals in Biology” (Pryor, W.A., Ed.) V, 29-64, Academic Press, New York. Corey, E.J. (1987) Pure and Appl. Chem. 59, 269-278. Galey, J.B., Bombard, S., Chopard, C., Girerd, J.J., Lederer, F., Do-Cao Thang, N’guyen Hoang Nam, Mansuy, D., and Chottard, J.C. Biochemistry, in press. Blackwell, G.J., and Flower, R.J. (1978) Prostaglandins 16, 417-425. Higgs, G.A., Flower, R.J., and Vane, J.R. (1979) Biochem. Pharmacol. 28, 1959-1961. Bellamy, A.J., and Innes, D.I. (1982) J. Chem. Sot. Perkin Trans. 2, 1599-1603.

345

its

BW 755C.

(Orsay, for

(ii)

maintain

whether

with

REFERENCES 1. 2.

is

the

interact

Ll,

that

to

for to

of

This

phenidone

Sante

and

presence

Morgenstern-Badarau

Rhane-Poulenc

site

inhibitors.

to of

(i)

compounds

would

necessary effects

Ll

Moreover, state

inhibitory

and

by

13-HPOD. active

state

(17).

EPR

the

by

substrates

in

clear

either

the

functions

are

already

be

being

the

NDGA

that

is

should

Ll.

02,

ferrous

is

LlFe(II)

such as phenidone,

determine

between

oxidized

Our

various

factors it

by

(17).

to

to This

derivatives

without

two

13-HPOD.

exist

compounds

Fe(II1)

by

oxidation

necessary

“peroxidase-like”

detection

LlFe(III)

differences

towards This

reduce

containing

is

acetaminophen

inactive

the

For

phenidone the

catechols

pyrazoline

However,

important

to

anaerobic and

of

work

LlFe(II1).

instance,

its

compounds

Further

reactions

in

of

oxidations

in

the

that

involved

and

(19). of

able

(16)

also

indicate

for

oxidation

formation

Ll-dependent

acid

is

its

with

compounds

N-alkylhydroxylamines

that

alcohol

(20).

A priori,

reduce

corresponding

dehydrogenation

but

hydroperoxide

the substrate

the

alkylhydroperoxides

this

to

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

help

Vol. 151, No. 1, 1988

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

8. Dusza, J.P., Joseph, J.P., and Bernstein, S. (1983) Eur. Pat. Appl. EP 70,376, Chem. Abstr. (1983), 99, 5626n. 9. Bellamy, A.J., and Innes, D.I. (1983) J. Chem. Sot. Perkin Trans. 2, 179-182. 10. Lindeke, B., Cho, A.K., Thomas, T.L., and Michelson, L. (1973) Acta Pharm. Suet. 10, 493-506. 11. Butler, R.N., and O'Donohue, A.M. (1978) Tetrahedron Lett. 19, 275278 12. Ghiglieri-Bertz, C., Coquelet;C.,-Alazet, A., and Bonne; C. (1987) Eur. J. Med. Chem. 22, 147-152. 13. Gibian, M.J., and Gallaway, R.A. (1976) Biochemistry 15, 4209-4214. 14. Axelrod, B., Cheesbrough, T.M., and Laakso, S. (1981) in "Methods in Enzymology" (Lowenstein, J.M., Ed.) 71, 441-451, Academic Press, New York. 15. Lee, W.E., and Zahorian, E. (1968) Phot. Sci. Eng. 12, 251-262. 16. Clapp, C.H., Banerjee, A., and Rotenberg, S.A. (1985) Biochemistry 24, 1826-1830. 17. Kemal, C., Louis-Flamberg, P., Krupinski-Osen, R., and Shorter, A.L. (1987) Biochemistry 26, 7064-7072. 18. De Groot, J.J.M.C., Veldink, G.A., Vliegenthart, J.F.G., Boldingh, J ., Wever, R. and Van Gelder, B.F. (1975) Biochim. Biophys. Acta 377, 71-79. 19. Marnett, L.J., Weller, P., and Battista, J.R. (1986) in "Cytochrome P-450" (Ortiz de Montellano, P.R., Ed.) 29-76, Plenum Press, New York. 20. Marnett, L.J., Siedlik, P.H., and Fung, L.W.M. (1982) J. Biol. Chem. 257, 6957-6964. 21. Markey, C.M., Alward, A., Weller, P.E., and Marnett, L.J. (1987) J. Biol. Chem. 262, 6266-6279. 22. Verhagen, J., Veldink, G.A., Vliegenthart, J.F.G., and Boldingh, J. (1979) in "Advances in the Biochemistry and Physiology of Plant Lipids", (Appelquist, L.A., and Liljenberg, C., Eds.), 231-236, Elsevier North Holland, Amsterdam.

346