Prostaglandin H synthase: An example of enzymic symbiosis

Vol.

130.

July

31,

No. 2, 1985

8lOCHEMlCALAND8lOPHYSlCALRESEARCH

COMMUNICATIONS

1985

Pages

PROSTAGLANDIN Richard

J.

Department

Received

Kulmacz,

of

June

H SYNTHASE: James

Biological

AN EXAMPLE F.

Miller,

OF

Jr.,

ENZYMIC

and

SYMBIOSIS

William

Chemistry, University Chicago, Illinois 60680

of

918-923

E.N.

Illinois

Lands at

Chicago

19, 1985

Reaction conditions which promote the heme-dependent peroxidase activity of prostaglandin H synthase appear to stimulate the heme-dependent cyclooxygenase activity also present in the synthase, even though the cyclooxygenase requires hydroperoxide for activity. However, aspirin-treated synthase, which retains only peroxidase activity, inhibited the cyclooxygenase activity of untreated synthase in the manner observed with similar levels of glutathione peroxidase. Any stimulatory effect of the synthase peroxidase on the synthase cyclooxygenase is thus likely to involve an intramolecular mechanism. Participation of peroxidase intermediates (Compounds 1 and II) in the initiation of a cyclooxygenase free radical chain reaction may provide an intramolecular mechanism for stimulation of the 0 1985 Academic Press, Inc. synthase cyclooxygenase by the synthase peroxidase.

Prostaglandin

H synthase,

biosynthesis

of

enzymatic

a fatty

forms

the

hydroperoxide,

oxide

for

initiation The is

of

synthase

in

the

value

for

synthase

PGG2 prevents

cannot

mechanism result

lower

this

1 Abbreviations

more

-6

of

peroxide

PGH

efficient

positive used

synthase

are:

PGG2.

that prostaglandin

$1.50

0 I985 by Academic Press, Inc. of reproduction in any form reserved.

918

of

the

the

importance

of

the

With activity

of

that

actually of

is

removes

activities

sufficiently

We propose

initiation symbiosis

levels

the the

of

a Km the

hydroperoxides,

but

to

suppress

the

the

presence

of

affords cyclooxygenase cyclooxygenase 62;

which

hydroper-

two

peroxidase

high

levels

activity. in

of

the two

activity

M lipid that

in

exhibits

biosynthesis.

the of

-8

these

eicosanoid

M (1.2).

step

prostacyclins,

activity

because

accumulation

peroxidase for

of

0006-291X/85

10

cyclooxygenase

heme-dependent

Copyrighr All rights

an

effectively

endogenous

control

about

10

between

committed

(cyclooxygenase)

requires

interest

first

and

a peroxidase

relationship

overall

the

oxygenase

and

and

considerable

of

acid

PGG2', (l),

hydroperoxides.

catalyzes

thromboxanes,

prostglandins,

activities:

synthase

which

PGH.

an

it

the

intramolecular The

reaction. activity prostaglandin

of

PGH H.

Vol.

130,

No. 2, 1985

synthase than

is other

BIOCHEMICAL

a much fatty

acid

heme-dependent

METHODS

more

explosive

BIOPHYSICAL

amplifier

oxygenases,

peroxidase

AND

AND

such

of as

RESEARCH

lipid

COMMUNICATIONS

hydroperoxide

soybean

levels

lipoxygenase,

which

lack

a

activity.

MATERIALS

Acetylsalicylic acid and N,N,N'.N'-tetramethylphenylenediamine were obtained from Sigma Chemical Co., St. Louis, MO. Arachidonic acid was purchased from Nu Chek Preps, Inc., Elysian, MN, and was treated with sodium borohydride to reduce any contaminating peroxides. Hydrogen peroxide was from Fisher Scientific Company, Fair Lawn, NJ. Prostaglandin H synthase was purified as the apoenzyme (3) and reconstituted with hematin (4). Cyclooxygenase activity was assayed with a polarographic oxygen electrode in a reaction cuvette containing 3 ml of 0.1 M potassium phosphate, pH 7.2/0.67 mM phenol/O.10 mM arachidonate (3). Peroxidase activity was assayed with hydrogen peroxide as substrate and tetramethylphenylenediamine as cosubstrate (1). Protein was assayed as described by Peterson (5).

RESULTS

AND

The

cyclooxygenase-catalyzed

reactions,

appears

having for

DISCUSSION

a free the

to

radical

of

the

However,

several

synthase

has

radical

observations

activity as

by

similar The

peroxidase

action

as

of of

the

one

synthase

dimer

with

the

cyanide

seem the

is

provide in

the

peroxidase

of

H202

parallels

cyclooxygenase

cyclooxygenase

are

the and their

reaction

initiation

PGH

activity. increase

closely

of

first. of

cyclooxygenase

(1)

in enzyme

activity

effectiveness

the

same at

in

essential

a route

cosubstrates

substrates

oxygenase acids

paradoxical

protein's

relative

of

to

presence

peroxidase

initiators

(9);

interfered

and with

(3).

heme-containing reactions

process

the

many b)

and

cyclooxygenase-catalyzed intramolecular

a)

activity

levels

on

peroxidase

effectivenesss

both

that

(7.8);

hydroperoxides

c)

indicated effect

include:

cyclooxygenase

might

fatty

polyunsaturated

seems

The

activity

known

Hydroperoxide

it

reaction.

peroxidase

a stimulatory

and

all of

mechanism.

(1.6).

chain

like

(6).

autooxidation

reaction

reaction

observations

relative

the

chain

a peroxide-removing

lipid

resemble

cyclooxygenase

initiation

These

reaction

or

by the

peroxidase could

intermolecular cyclooxygenase

in

conceivably interactions on

919

another

stimulating occur

by of

dimer.

the

an peroxidase Possible

on

Vol.

130,

No. 2, 1985

intermolecular

processes

dimers,

production

(10)

would

of

AND

include

a diffusible

BIOPHYSICAL

direct

RESEARCH

contact

stimulant,

or

COMMUNICATIONS

between

removal

synthase

of

a toxic

byproduct

* To

determine

if

intermolecular of

BIOCHEMICAL

in

adding

only

synthase,

to

treated 1% of that

its

(2

aspirin

synthase

was

as

aspirin

would

of not

the

peroxidase

(1.1

the

JIM)

significantly

mol

Aspirin-Treated moluntreated

to

due

to

the

synthase

because during

decreased

in

untreated the

additional

and

not

such the

to

the

a level 30

Synthase Synthase

Fiqure 1: Inhibition of cyclooxygenase activity of PGH synthase by aspirin-treated PGH synthase. The cyclooxygenase activities of the indicated proportions of aspirin-treated synthase and untreated synthase 1.1 nM subunit) were assayed as described in the text. 920

the

Methods.

synthase)

it,

of

and

and

synthase with

90%

injected,

Materials

was

below

cuvette

synthase

along

inactivate

at

was

aspirin-treated

added

to

aspirin-treated

untreated

decrease

was

activity

nM subunit)

the

The

holoenzyme

reaction

aspirin-treated 1).

aspirin-treated

this

in

be activity

activity of

described (of

of

of (~50

cyclooxygenase

amounts

as

of

synthase

a cyclooxygenase

(Figure

aspirin

PGH

could

cyclooxygenase

form

the

Various

activity

activity

peroxidase on

the

Pure

synthase

ratio

the

effect in

leaving

determined

the

increased

the

reduce

into

untreated

peroxide-scavenging amount

to

while

cyclooxygenase fashion

small

mM)

activity

a linear

of

activity,

injected

before

observed

examined

treatment.

then

cyclooxygenase

effect

PGH synthase.

level

synthase'were

The

intact

initial

immediately

we

peroxidase

aspirin

before

stimulatory

nature,

the some

with

the

set

of

Vol.

130,

No. 2, 1985

used

to

assay

the

x-axis

mole

of

cyclooxygenase indicated

of

corresponded reasonably

(in

as

form

of

glutathione

are

interfere

The

with,

activity the

same

of of

did

of

untreated

when

the

than

augment,

need

be present

10 40

(1).

ratio

of

70

peroxidase not

stimulate, synthase,

evidence

therfore

points

peroxidase

dimer

peroxidases

per

about

synthase

synthase)

holoenzyme

heme-containing

rather

adding

only

to

cyclooxygenase

ratio

accumulated

curve

protein

ratio

activity

did.

in

the

aspirin-treated

cyclooxygenase

Other

cyclooxygenase.

Thus,

cyclooxygenase

present

treated

protein/protein

(1).

peroxidase of

of

the

peroxidase/cyclooxygenase

the

the

of

activity

glutathione

suppressed

intermediates

10 moles suppress

This

cyclooxygenase

the

a stimulation

to

the

the

instead

much to

to

suppressed

but

about

maximally

enzyme.

COMMUNICATIONS

Extrapolation

a peroxidase/cyclooxygenase

close

separately

of

could

untreated

to

(11).

a ratio

protein

the

BIOPHYSICALRESEARCH

activity

that

untreated

activity

that

BIOCHEMICALAND

with

of

the

a cell

initiation

of

would

also

tend

prostaglandin

formation. Because

only

catalytic

one

activity

heme (4).

it

seems

cyclooxygenase

by

the

peroxidase

cyclooxygenase

reactions

share

One 2,

where

in

the

possible

scheme

peroxidase of

mechanism

each

molecule

of

activating

another

reaction.

With

molecule

of

would

be

much

more

one

molecule

postulated

the

PGG2

such

as

than

that

hydroperoxide soybean

of

synthase

dimer another

(13),

921

proposed

Figure to

each

character be

of

which

(12)

of

the

accounting

of

chain a

action

acceleration

initiation

as

capable

involves

cyclooxygenase

which

serve

This

would

Such

perhaps

in

cyclooxygenase

during

initiation

shown

reaction.

hydroperoxide.

the

are

branched

in

forms.

is

II,

process,

a system

and

enzyme

I and

initiating

to

for lipoxygenase

one

the

peroxidase

intermediates

acceleration

regard

the

autoaccelerative,

a two-step

the in

of

cyclooxygenase

of

radical-chain

by

full

intermediate

Compounds

an

for

stimulation

common

sharing

dimer.

initiation

explosive

for

because

synthesized

synthase

order

of

occurs some

have

hydroperoxide,

second

that

cyclooxygenase

would

dimer

likely

intermediates,

initiation

reaction

for

per

would

required reaction,

such for

the

only as observed

be

Vol.

130,

BIOCHEMICALAND

No. 2, 1985

ROOH ROH

BIOPHYSICAL

ROOH

RESEARCH

COMMUNICATIONS

q ROOH

AH

/

Figure 2: A hypothetical mechanism for the stimulation of synthase cyclooxygenase activity by synthase peroxidase intermediates. The formal oxidation states of the heme iron of the synthase (E) are indicated by Roman numerals. ROOH: Lipid hydroperoxide; ROH: lipid alcohol; ROO: hydroperoxy radical; RH: polyunsaturated fatty acid; AH2: peroxidase cosubstrate; AH: oxidized cosubstrate.

differences

in

enzyme. via

kinetics

Alternatively,

Compounds

small

The

the

I or

amounts

oxygenase

of

II

retention in

explosive

the this

same

that survival

ACKNOWLEDGEMENT: Public Health the University

the

be

cyclooxygenase

much

than

the

non-heme

of

for

soybean

symbiotic

many

rapid

many

animals

the

soybean

cyclooxygenase sensitive

to of

of

two

these

suggests

two that

by

low

activities

catalyt the

sensi

amounts

provides

C

may

have

species.

This work was supported by Service (GM30509) and a Medical of Illinois at Chicago, College

3. 4. 5. 6.

Kulmacz, R-J., Lands, Ohki, S., Ogino, N., 254, 829-836 Kulmacz, R-J., Lands, Kulmacz, R.J., Lands, Peterson, G.L. (1979) Hemler, M.E., Lands,

W.E.M. Yamamoto, W.E.M. W.E.M. Anal. W.E.M.

tive

of

a grant from the United Scientist Fellowship of Medicine

States (JFM) from

REFERENCES 1. 2.

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oxygenase.

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and

the

initiation

linkage

prostaglandin

combination

more

of

peroxide-dependent

iron

in

and

initiation

inherently

holoenzyme

value

the

peroxide-dependent

unusual

enhancement

hydroperoxide important

in of

the

may

between

hydroperoxide

reaction

activities and

reaction

(1983) S., (1985) (1984) Biochem. (1980)

922

Prostaglandins Hayaishi. 0.

25, (1979)

Prostaglandins

J. Biol. J.

100, Biol.

Chem. 201-220. Chem.

29, 259, 255,

531-540. J. Biol. 175-190. 6358-6363. 6253-6261.

Chem.

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1. 8. 9. 10. 11. 12. 13.

BIOCHEMICAL

AND BIOPHYSICALRESEARCH

COMMUNICATIONS

Nugteren, D.H., Beerthuis, R.K., van Dorp, D.A. (1966) Rec. Trav. Chim. Pays-Bas 85, 405-419. Takeguchi, C., Kohno, E., Sih, C.J. (1971) Biochemistry 10, 2372-2376. Marshall, P.J., Kulmacz. R.J., Lands, W.E.M. (1984) in Oxygen Radicals in Chemistry and Biology (W. Bors, M. Saran, D. Tait, eds.), pp. 299-304, de Gruyter, Berlin. Egan, R.W., Paxton, J., Kuehl, F.A., Jr. (1976) J. Biol. Chem. 251, 7329-7335. Rome, L.H., Lands, W.E.M. (1975) Proc. Natl. Acad. Sci. USA 72, 4863-4865. Boudart, M. (1968) Kinetics of Chemical Processes, pp. 124-136, Prentice-Hall, Englewood Cliffs, NJ. (1972) J. Biol. Chem. 247, 1038-1047. Smith, W.L., Lands, W.E.M.

923