Tight binding of oxaloacetate to succinate dehydrogenase

Tight binding of oxaloacetate to succinate dehydrogenase

Vol. 51, No. 4, 1973 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TIGHT BINDING OF OXALOACETATETO SUCCINATEDEHYDROGENASE Anna Priegnits, Ne...

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Vol. 51, No. 4, 1973

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

TIGHT BINDING OF OXALOACETATETO SUCCINATEDEHYDROGENASE Anna Priegnits, Nencki Institute

Received

February

Olga N. BrehevskayaX and Lech Wojtcsakxx of Experimental

Biology,

22,

Warsaw

Poland

26,1973

SUMMARY: [14C]Oxaloacetate forms a stable complex with succinate dehydrogenase which withstands repeated Sephadex filtration. Oxidized glutathione, 2-thenoyltrifluoroacetoae, KCN and ageing at +4’ at neutral pH do not prevent the enzyme to bind oxaloacetate. The binding is prevented by succinate or malonate but the complex, once formed, can not be split by these compounds, although the enzyme activity can be restored: the reconstitutive property of succinate dehydrogenase is, however, irreversibly lost. Bound oxaloacetate does not exchange with added oxaloacetate, but can be released by perchloric acid. Sonic particles of beef heart mitochondria can also bind oxaloacetate. However, this complex can be split by succinate or malonate. It

has been shown (1 -

other competitive that

inhibitors

the inhibition

the inhibitor. substrate

The reversal

oxaloacetate

of succinate

develops slowly

(OA)

complex with OA;

upon incubation

of the inhibition

by high concentrations

complex which could not be split

tinent

published to this

possibility

subject

of a stable

filtration,

similar

(6)

reported

preparations

conformational

of a relatively

stable

These results

Recently,

two reports

et al.

described by ourselves

OA the presence of tightly

(4);

bound OA

of SD. IA the present paper we describe of Biophysics,

(5)

1034

were per-

showed the to Sephadex

and Keamey et

ir,

deactivated

in

more detail

Academy of Sciences of

COrrespOAdeACe should be addressed.

Copyright 0 19 73 by Academic Press, Inc. All rights of reproduction in any form reserved.

in-

the reaction

complex between SD and OA, resistant

to that

axpla-

we studied

have appeared. Vlnogradov

x Permanent address: Institute USSR, Moscow, USSR. xx To whom all

form (4).

of

1. SD forms a stable

by gel filtration.

IA a preliminary

formation

al.

Two alternative

2. OA induces a slow but reversible

between [14C]OA and SD and found the formation

already

of the enzyme with

have been proposed (2):

of SD. To check the first

activation

from

differs

dehydrogenase (SD) by the fact

or by removal of OA was also slow (2).

of this peculiarity

nations

that

3)

our

Vol.51,No.4,1973

BIOCHEMICAL

study on complex formation the properties

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

between SD and OA and present further

complex. We also provide

of this

on the enzyme molecule Involved for the reconstitutlon.of

in the binding

the succinate

data on

an evidence that

a site

of OA 1s also responsible

oxidase system (7, 8).

MATERIALS ANDMETHODS Crude soluble, heart mltochondrla

reconstltutlvely

active,

SD was obtained from beef

by the method of King (9) with slight

modlflcatlons

(2) and passed through a column of Sephadex G 25, equilibrated Trls-HCl

+ 1 mMEDTA (pH 7.4),

was prepared according omitting

the last

to remove succlnate.

to Dervartanlan

was determined spectrophotometrlcally 2,6-dlchlorophenollndophenol obtained

from uniformly

79 mCl/mmol) by Incubating -oxaloacetate formation

purified

Enzyme activity

with phenazlne methosulfate

[14C]aspartate

before

(2).

(specific

The efficiency

exceeded 7% and the remaining

[14C]aspartate

and

[14C]OA was

activity

with an excess of 2-oxoglutarate

transaminase (EC 2.6.1.10).

SD

occasionally

precipitation.

(111, as described

labelled

Partially

and Veeger (lo),

(2nd) ammoniumsulfate

with 10 mM

and glutamate of

[‘4C]OA

was not removed.

[14C]OA

thus formed was Incubated with SD during 30 mln at 20’.

mixture

was then cooled to 0’ and passed through a column (2.2 x 20 cm)

of Sephadex G 25 equilibrated SD eluted activity

with 10 mMTrls-HCl

+ 1 mMEDTA (pH 7.4).

In the void volume of the column was collected and radioactivity

determined according

were measured. Peptide-bound

to Cerlettl

et al.

the

and the enzgmic flavin

was

(12).

RESULTS When SD was prelncubated

with

[14C]OA

and the mixture

nated on Sephadex two peaks of radioactivity coinciding

with the proteln

to low molecular easily

and SD activity

weight constituents

be measured in the effluent,

since the activity

was restored

(Fig. in spite

were found, the first

one

and the second corresponding 1). The enzyme activity of pretreatment

by high concentration

1035

was fractlo-

with

of succinate

could OA,

in

Vol. 51, No. 4,1973

BIOCHEMICAL

AND BIOPHYSICAL

Fraction

RESEARCH COMMUNICATIONS

No

Fig.

1. Formation of a stable complex between oxaloacetate and succinate recipitation purified SD (last ammoniumsulfate 10 mM Tris-DC1 + 1 mMEDTA (pH 7.4 7 with 40 )Gl OA and 2.5 ml of the mixture was subjected to Sephadex ffltration. Fractions of 1.0 ml were collected and examined for .SD activity (o- - -01 and radioactivity (-1. SD aotivity is expressed as change in the absorbance at 600 nm in the assay system per min per ml of the effluent.

the assay medium (2). [14C]malonate

When [14C]OA was replaced

Sephadex filtration

the enzyme and the radioactivity The elution

of a part

resulted hot

by [14CJaspartate

in a complete separation

of [14cJo~ in the void volume of the column

but not with human r-globuline

heart mitochondria. jected

When the effluent

nor the matrix containing

to a second Sephadex filtration,

again eluted

of

shown).

was observed with both the crude as well as the purified tions,

or

fraction

of beef

SD-OA complex was sub-

practically

in the void volume. This procedure

enzyme prepara-

all

radioactivity

could be repeated

was several

times with the acme result. To get more information factors

known to interact

an oxidizing

on the site

of binding,

with SD was studied.

agent for protein

the effect

Oxidized

glutathione

SH groups and has been reported

1036

of some is

to prevent

Vol. 51, No. 4, 1973

BIOCHEMICAL

the reconstitution

agent.

SD (14).

with sulfhydryl

SD from the respiratory

bind to non-heme iron, chelating

The latter

particle-bound,

of SD with either

of

most of the enzyme activity,

glutathione

on the binding

had little

on

OA

abolished

the binding

was largely

added after

preincubation

incubation since it under

of succinate

(not shown) was present

complex was already

[14C]OA.

somewhat effect.

binding.

However,

diminished

or

the binding.

When high concentration lonate

or

in the loss of

pH, resulting

effect

it

was without

ageing at 30’ and pH 9.5 during 2 to 3 hours strongly completely

of

In some experiments

19 hours at neutral

for

but not soluble,

whereas in others it

OA

a known iron-

‘50 mMoxidized

had no effect

of 2 mMKCN was less clear.

diminished the binding

known

chain (7, 13, 14). Cyanida can also

inhibits

0.2 mMthenoyltrifluoroacetone

Ageing of SD at +4’

groups and is

as does 2-thenoyltrifluoroacetone,

Pretreatment

The effect

oxidase system from soluble SD (7).

of the succinate

Also cyanide may slowly react to dissociate

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(Table I) or,

during the incubation

diminished.

However, if

even better,

of SD with

succinate

of the enzyme with OA, i.e.

is known (2) that

This observation the inhibition

[14C.]OA

or malonate was

when the SD-OA

formed, bound OA was not released even after

at 20’ (Table I).

ma-

is of particular

is almost completely

10 min importance released

these conditions.

When the complex of [14C]OA with SD was incubated with unlabelled OA and passed through Sephadex thereafter,

was recovered

in the fraction

containing

once bound to SD, is not easily

more than 80$ radioactivity the enzyme, indicating

exchangeable with external

could be, however, released when the enzyme ric

acid.

About 9OJ6radioactivity

and was tentatively and extraction

identified

(16) as a mixture

was formed apparently analytical

was precipitated

was then released into by means of thin of oxaloacetate

by non-enzymatic

procedures. 1037

OA.

layer

OA,

Bound

OA

with perchlo-

the supernatant

chromatography

and pyruvate.

decarboxglation

that

of

OA

(15)

The latter during the

Vol. 5 1, No. 4, 1973

BIOCHEMICAL

AND BIOPHYSKAL

RESEARCH COMMUNICATIONS

Table I. Effect of succinate on the binding of oxaloacetate to succinate dehydrogenase. Preparations of partially purified SD (last ammonium sulfate precipitation omitted) were incubated with [14C]OAT Succinate was added either from the beginning of incubation (Expt. li or after 20 min and the incubation continued for next 10 min (Expt. 2). The mixture was then fractionated on Sephadex column. bound to SD

[14C]OA

Expt. No.

Additions

1.

to the incubation

($ of total

radioactivity)

None

12.0

40 mMsuccinate

2.

present from the beginning of incubation

2.4

None

9.7

40 mM succinate

A possibility

added after

that

20 min

OA forms a Schiff

SD-OA complex was therefore

treated

acid thereafter.

into

exactly

that no Schiff The ratio containing specific OAfflavin

base with SD was checked. The

with borohydride

tated with perchloric the supernatant,

8.4

The radioactivity

as without

borohydride

was released treatment,

of OA to peptide-bound

flavin

was determined in fractions

SD-OA complex. The amount of OA was calculated radioactivity ratio

of [14C]aspartate.

were obtained

beef heart mitochondria centrifugation.

showing

base was formed.

The following

in three experiments:

To check whether OA can bind to particulaCe

It

were incubated

was found (Table II)

particles

whereas [14C]aspartate

destroyed

by alkali

Contrary

(17) and precipi-

treatment

was not.

values for

the

1.3, 0.7 and 0.7.

SD, sonic particles

with [14C]OA that

from the

and separated by

[14C]O~ was taken up by the

Particles

(18) lost most of their

in which SD had been ability

to bind Oh.

to what was observed with soluble SD, OA bound to particulate

SD could be released by subsequent addition

of succinate

or malonate.

DISCUSSION The presence of minute amounts of OA in some preparations

1038

irom

of sol-

BIOCHEMICAL

Vol. 51, No. 4, 1973

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Table II. Binding of rllc Cloxaloacetate to submitochondrial narticles. Sonic particles from beef heart m tochondria (7 mg protein/ml) were incubated JO min at 20’ with 8 )@4[1 t C]OA in the medium containing 120 mM KC1 + 10 mMTris-HCl (pH 7.4), separated by centrifugation at 100,000 x g during 45 min and washed twice. SD-free particles were obtained by incubating sonic particles at pH 5 during 2.5 hours under aerobic conditions (18). In . samples with [ 9 ‘%]aspartate, glutamate-oxaloacetate transd E-oxoglutarate was replaced by glutamate in the ~:~t~g~~~r~~:~~"~l~,OA.

Additions

Radioactivity bound to the particles (counts/min/mg protein)

to the incubation

Sonic containing B-w SD ---w-w particles -----------------(control)

[14C]OA

[14C]OA + 40 mEImalonate present from the beginning of incubation [ 14C]OA

+ 40 mMmalonate added after

10 min

[ 14C]Aspartate SD-free particles ----------w---s 460

[14C]OA

[ 14C]Aspartate

uble SD has already et al.

(5).

sufficient

35

been described

Kearney et al. experimental

(2).

Recent observations

(6) and of the present

evidence for

vin,

found in the present study,

et al.

investigation

the existence

between SD and OA. The lower value of 0.7 for

provide

of a stable

the ratio

is close to that

of Vinogradov

complex

of OA to SD fla-

observed by Vinogradov

and somewhat higher than the value of 0.5 found by Kearney

The difference

may be due to the fact

formed by the enzyme itself measured the binding activity

of uniformly

partial

decarboxylation,

estimated.

Therefore,

that Kearneg et al.

whereas Vinogradov

et al.

et al.

determined OA

and ourselves

of added OA. The amount of OA measured by the radfo-

labelled and it

[14C]OA

may be underestimated

seems likely

that

SD flavin

the true value of OA to flavin

1039

ratio

due to a may be over-

in the stable

Vol. 51, No. 4, 1973

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

from added OA may be even 1:l

formed

complex

BIOCHEMICAL

suggested by Vinogradov Xearney et al.

et al.

(5).

(6) was slowly

or higher.

This is also

The SD-OA complex described by

split

by high concentrations

and malonate vhereas in Vinogradov’s

of succinate

and our experiments

be released from the complex by the substrate formed. This may be due to a different

OA could not

or malonate once it was

proportions

of OA and SD in the

complexes. The site gradov et al. hydryl

and mode of binding (5) postulate

of OA to SD is not quite

the formation

of thiosemiacetal

group of the enzyme. However, in their

prevented

only when all

titration.

After

altered

be affected.

The fact

protein

(cf.

on

treatment

was

the enzyme conformation binding sites

the complex can be split

the binding

must nay also

by denaturing

acid should also be taken into

the

consideration

mechanism. The present experiments

cyanide and thenoyltrifluoroacetone binding

with a sulf-

hands the binding

19) and other potential

that

with perchloric

when speculating

Vine-

25 SH groups of SD (19) were blocked by Ag'

such extensive

be seriously

clear,

with

exclude non-heme iron as a possible

site.

OA bound to soluble

whereas the catalytic conditions different

(2).

SD can not be released by ouccinate

activity

could be fully

This indicates

from the catalytic

that center

the tight

restored binding

the enzyme.

of

or malonate

under appriopriate occurs at a site

However,

the ability

of SD to bind to membrane fragments devoid of SD and to reconstitute succinate

oxidase is irreversibly

gests that

for

OA binds to a site

the reconstitution.

is less tightly therefrom It

for

lost on

after

(2).

This sug-

the enzyme molecule which is responsible

This is also supported by the observation

bound to [email protected]

by an excess of succinate can be therefore

OA treatment

and can be removed

or malonate.

concluded that

OA. One of them is the catalytic

particles

that OA

SD posseses two affinity

site

1040

sites

of the enzyme which *recognizes*

BIOCHEMICAL

Vol. 51, No. 4,1973

OA as substrate

analogue

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and reacts vlth

second one is most likely

a site

nate oxidage and can

a stable

A

mational

tight

binding

form

of

OA

involved

it

in a competitive in the reconstitution

does not exclude,

(2) and recently

The of succi-

complex with OA. of course, that

change of the enzyme occurs concomitantly,

by ourselves

ray.

as already

shoun by Eearney et al.

a confor-

suggested

(6).

REFERENCES 1. 2. 3. 4. 5. 6. 2 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Chappoll, J.B. (1961) in Biological Structure and Function (Goodwin, T.V. and Lindberg, 06, eds) vol. 2, pp. 71 - 83, Academic Press, London and New York. Wojtczak, L., Wojtczak, A.B. and Ernster, L. (1969) Biochim. Biophgs. Acta, 191, 10 - 21. Zeylemaker, W.P., Klaasse, A.D. and Slater, E.C. (1969) Bioohim. Biophys. Acta, 191, 229 - 238. Priegnitz, A. and Wojtczak, L. (1970) 8th Meeting of the Polish Biochemical Society, Szczecin, Abstracts, p. 27. (in Polish). Vinogradov, A. D., Winter, D. and King, T.E. (1972) Biochem. Biophys. Res. Commun.49, 441 - 444. Kearneg, E.B., Ackrell, B.A.C. and Mayr, M. (1972) Biochem. Biophys. Res. Commun. 49, 1115 - 1121. T.E. (1960) Proc. Roy. Sot. B, 152, 163 - 187. Biol. Chem. 238, 4037 - 4051. Biophys. Acta, 58, 375 - 377. Dervartanian, D.V. and Veeger, C. (1964) Biochim. Biophgs. Acta, - 247 %g23’?.E (1963) J. Biol. Chem. 238 4032 - 4036. Cerlitti, ‘P., Strom, R. and Giordano: M.G. (1963) Arch. Biochem. Biophys. 101, 423 - 428. Wilson, D.F. and King, T.E. (1964) Biochim. Biophys. Acta, 92, 173 ::$ T.E (1966) Adv. Enzymol. 28 155 - 236. Hyeri, W.;. and Huang K.Y. (1966)‘Analyt. Biochem. 17, 210 - 213. Friedmann, T.E. (1957) in Methods in Enzymology (Colowick, S.P. and Kaplan, N. 0.) eds) vol. 3, pp. 414 - 418, Academic Press, New York. Lai, C.Y., Tchola, 0.) Cheng, T. and Rorecker, B.L. (1965) J. Biol. Chem. 240, 1347 - 1350. King, T.E. (1961) J. Biol. Chem. 236, 2342 - 2346. Pagani, S. and Cerletti, P. (1972) Abstr. Commun.Meet. Fed. Europ. Biochem. Sot. 8, Abstract lo. 444.

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