Localization of the oxaloacetate-binding site in the iron-flavoprotein subunit of the inhibitor-succinate dehydrogenase complex

Localization of the oxaloacetate-binding site in the iron-flavoprotein subunit of the inhibitor-succinate dehydrogenase complex

Vol. 56, No. 2, 1974 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS LOCALIZATION OF THE OXALOACETATE-BINDING SITE IN THE IRON-FLAVOPROTEIN ...

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Vol. 56, No. 2, 1974

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

LOCALIZATION

OF THE OXALOACETATE-BINDING

SITE IN THE IRON-FLAVOPROTEIN

SUBUNIT OF THE INHIBITOR-SUCCINATE

DEHYDROGENASE COMPLEX

Daryl

B. Winter and Tsoo E. King Department of Chemistry State University of New York Albany, New York 12222

Received

November

26,1973

SUMMARY--The oxaloacetate in the inhibitor-succinate dehydrogenase complex could be displaced by the substrate or inhibitors. The oxaloacetatebinding site is found in the iron-flavoprotein subunit of the enzyme by two methods of dissociation of succinate dehydrogenase into subunits. It

was suggested

contains

at least

two moieties

spectroscopic

protein

sulfide, cow

two distinct

moieties

by low

titration

site

This

note

reports

is

in the

EXPERIMENTAL--The

(2)

as previously

amination

of the amino

presence

sulfate

group

for

subunit

14 C-labeled

of 14 C-labeled

of transaminase.

The gel

(SDS) and B-mercaptoethanol

binding

and related

of preparations

except

electron

these

paramagnetic

separation

A sulfhydryl

site

(SDH)*

Recently

(1).

temperature

(3).

iron-flavoprotein

(4)

dehydrogenase

and by the chemical

the direct

methods

described

group, with

of an

not

labile

oxaloacetate

evidence

that

the OAA

of succinate

dehydrogenase.

and determinations

were

OAA was prepared

by trans-

asparate

to n-ketoglutarate

electrophoresis

was performed

in sodium according

in

dodecyl-

to Weber and

(5). RESULTS AND DISCUSSION--In

was bound

to soluble

be subjected binding

iron-protein

and an iron-flavoprotein

(4).

Osborn

succinate

of SDH has been shown as the

binding

the

ago that

have been demonstrated

resonance iron

some time

*Abbreviations:

our

previous

SDH and the enzyme-inhibitor

to purification

was effected

confirming

by Sephadex

by incubation

results

complex

chromatography.

of OAA and the

OAA, oxaloacetate; SDS, sodium succinate dehydrogenase

thus

OAA

formed

However,

dehydrogenase

dodecylsulfate;

(4),

could

the maximal

for SDH,

a few

Vol.

56, No.

minutes

2, 1974

BIOCHEMICAL

at. 38".

The ratio

enzyme was again than

that

extracted

found

recently from

We previously through conclusion (8)

is

and their

acetal

could

to be about

0.7.

by Kearney

powder

reported

a two stage

that

reaction

However,

r'r

-----n-IT

25

5

16

IO

significantly

the succinate

of Wojtczak

and inhibitors

form Our

(7) and Nicholls in a thiosemi-

as shown in Fig.

1.

Numixr

--

15

20

25

-'-ET;-

5

IO

15

--cl

tn

20

Fumarate

25

!

16

z 0C-l 16”.? 12 2 0Fa 4

04

1.

dehydrogenase

enzyme and oxaloacetate.

0.0

Fig.

higher

in a thiosemiacetal

E 0 x +i 1.6 Lt 1.2

0

in the

(6). OAA is

Succinote

,=tWSDi-!Dt~

is

COMMUNICATIONS

flavin

even the OAA incorporated

by substrate

20

for

reports

Fraction

15

ratio

the

cocorkers.

IO

This

the bound

by the recent

be displaced

nonextractable

heart

between

RESEARCH

acid

--et al.

of beef

supported

5

BIOPHYSICAL

of OAA to the

reported

acetone

AND

15

25

35

15

25

35

Elution

Volume

(ml)

15

25

35

0

14 C-labeled The purified OAA-succinate dehydrogenase complex, 10 mg/ml, was incubated with phosphate, 50 mM; succinate 20 mM; fumarate 20 mM; malonate, 20 mM; oxaloacetate, 20 mM; or B-mercaptoethanol, 15 mM; for 10 min at 38". The mixture was then rapidly cooled to 4' and chromatographed on a Sephadex G-50 column with The continuous 50 mM phosphate buffer, pH 7.4, anaerobically. line is from the automatic recording of transmittance (%T) at 280 nm for protein, the open circles are the protein content per fraction by the biuret method, and the solid circles are the radioactivity (CPM) per vial by a liquid scintillation method. Roman numerals I and II are the peak numbers. Recoveries for radioactivity and protein were greater than 92%.

291

Vol.

56, No.

2, 1974

Phosphate other

exhibited

hand,

displaced from

BIOCHEMICAL

70,

(not

60,

inversely complex

related

of Ki is

magnitude

to release

labeled),

complex.

or the

in the order < Kyarate

(1.2

x 10 -3 M (10))

is much larger

14 C-OAA was only

constant;

complex

with

separation

of the subunits in Fig.

This

thiol,

with

succinate

These

facts

formed

for are

between

is not

the practical

the

inhibitor.

enzyme but rather

in line

with

our

with

conclusion

a complex

effect

of a

application

of

electrophoresis.

70% of the bound It

does not

that

OAA.

compete

the enzyme sulfhydryl (4)

complex

enzyme-inhibitor

medium by SDS gel

a competitive

fact

methods.

and for

about

This

Km is

OAA in the

displaced

succinate

succinate-SDH

the competitive

mercaptoethanol

OAA.

whereas

to test

in dissociation

the behavior for

the

of E-mercaptoethanol localization

dehydrogenase,

medium was still l4 C-labeled

thiosemiacetal

for

for

of the

the

succinate Km

capability

by simple

in

< Kylonate

Although

constant

to displace

in order

known that

M (9))

to that

constant

was,

OAA

of the substrate-

is

x lO+j

inferior

be determined

C-labeled

for

a thiosemiacetal

OAA. is

OAA and the enzyme.

experiments

of succinate

the

lF,

however,

Although our

the

It

OAA the Ki

dissociation

examined

sulfhydryl

(1.5

equilibrium

of mercaptoethanol

was also

As shown

slightly

and cannot

The ability

constant

on the

and succinate 14

of displacement

(2 x 10 -3 M (11)).

a true

no true

has been reported

fumarate

complex.

than

COMMUNICATIONS

OAA, while

of the

The degree

of K.yM

x 10 -5 M (lo))

kinetic

malonate,

inhibitor-enzyme

may be due to Ki being

RESEARCH

any bound

to the dissociation

(4.5

to release

BIOPHYSICAL

25, and 60%, respectively,

inhibitor-enzyme

general, enzyme

ability

oxaloacetate about

the

little

AND

a useful OAA-enzyme

for

complex

in SDS and b-mercaptoethanol

(5).

of radioactivity

is

and protein

of the O&A binding

the SDS gel tool

complicated

electrophoresis

our

probe.

shown

in Fig.

292

site

in

Consequently

the subunit

gel

we subjected electrophoresis

of the distribution 2.

of

in a dissociating

to the conventional The results

the design

The first

major

pattern protein

Vol. 56, No. 2, 1974

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

CPM

-300

- 200

i - 100

/I 0.0

2

3

4

5 gel

Fig.

2.

Slice

8

9

IO

II

lt

4 0

2

Number

The distribution of protein and radioactivity in the subunit fractions of the inhibitor-enzyme complex. Approximately 3 mg of a purified 14C-labeled OAA-SDH complex was incubated 5 min in a mixture containing 0.01% SDS, 0.005% B-mercaptoethanol and 50 mM phosphate buffer, pH 7.4. The mixture was separated by large scale gel electrophoresis adapted from the method of Weber and Osborn at 40 milli-amphere. The gel was uniformly sliced at approximately 5 mm thickness. The gel slice was solubilized in an "NCS Solubilizer" (purchased from Amersham/Searle) and counted for radioThe radioactivity is activity in a liquid scintillation counter. The gel was also presented in bars using the left ordinate. stained by Coomassie Blue and the stained gel was scanned at a wavelength of 600 nm as represented by the continuous solid curve. The first protein peak (in slice 4) was the iron-flavoprotein subunit (Fp) of 70,000, the second protein peak (in slice 6) was an impurity which was substantiated from evidence of other and the last protein peak (in slices experiments (to be published); 9-10) was the iron protein subunit (Ip) of 27,000. The dye front is not shown. The initial blue stain and radioactivity was due to the denatured polymerized aggregate impermeable to the gel, probably resulting from the extremely low mercaptoethanol concentrations employed.

peak equivalent

to a molecular

subunit

last

and the

of the

dehydrogenase.

above

the background,

in slice

7

6

number

protein It

weight

of 70,000*

peak of 27,000X is

clear

radioactivity

only

from a "standard" weights.

293

iron-flavoprotein

iron

protein

peak possessed

to the protein.

1 was due to some denatured

*These figures were obtained proteins of known molecular

was the

the first

bound

was the

polymerized curve

subunit significant,

The radioactivity aggregate

which

consisted

which of

Vol.

56, No. 2, 1974

did

not

penetrate

the

of the released Because without gel

BIOCHEMICAL

free

gel

inherent

dissociating

the

acid

58 mg protein, mixture

fractions. protein

was mixed

It

whereas

with

the

tail

end

by the SDS-mercaptoethanol

another

method.

centrifuged

complex,

and then

to separate washing

only

of the Radioactivity Iron-Flavoprotein

the

acid

contained

475 cpm/mg.

Subunits

this

at pH 7.8. cycle

supernatant

and of the

(Fo)

of trichloro-

5.0 ml containing

thawed;

the pellet

showed

The subunits

by the action

incompletely,

nitrogen

the supernatant

and the

COMMUNICATIONS

the two subunits

1.0 ml 4 M trichloroacetic

appropriate

Distribution

TABLE I.

12 was from

complex

of 14 C-OAA-enzyme

in liquid

was then After

RESEARCH

in separating

we sought

although

A sample

was frozen

3 times.

complication

as described,

(3).

in number

enzyme-inhibitor

of SDH can be separated, acetic

that

BIOPHYSICAL

OAA.

of the

electrophoresis

whereas

AND

This

Iron

was repeated

and the 1320

The

pellet

cpm per mg

experiment

Protein

(Ip)

in SDH and in Subunit

Fractions*

CPM/mg found Succinate

dehydrogenase

Supernatant Pellet

CPM/mg calculated

Fp:Ip 1:I

untreated

fraction

fraction

475

475

1320

1400

1:5 1:0.65

*The SDH as well as the supernatant and the pellet fractions was subjected to the gel electrophoresis in a medium containing SDS and b-mercaptoethanol. The Coomassie Blue-stained gel was scanned and the areas under the ironflavoprotein (Fp) (mol. wt. 70,000) and the iron protein (Ip) (27,000) bands re integrated and the ratio of Fp:Ip is presented in the table. The E C-labeled OAA-SDH complex was treated with TCA as described in the text. The supernatant and the pellet fractions were retained for 14C determination. The calculated value as shown in the last column was obtained by taking the figure 475 cpm/mg protein in the supernatant fraction as entirely due to Fp.

294

Vol.

56, No.

the

verifies that

the

conclusion

OAA binding

radioactivity the

iron

BiOCHEMICAL

2, 1974

protein

the

fraction iron

contamination

with

the extent did

observed

binding

is

indeed

which

in all

Consequently,

heavily

might

fractions

iron-flavoprotein

that

be

the contain

SDS-gel were

However,

is evident

might

also

employing

fractions.

only

Likewise,

As shown in Table

it

The

treatment,

probability experiments

in both

contamination,

electrophoresis,

contains

subunit.

and the supernatant

in the

ideally acid

flavoprotein

COMMUNICATIONS

subunit.

the trichloroacetic iron

gel

iron-flavoprotein

of contamination.

occur

RESEARCH

by direct

fraction,

flavoprotein)

of the pellet

made fo-c the site

the

subunit.

electrophoresis

in the

from

BIOPHYSICAL

obtained

supernatant

(iron

protein

to determine

is

subunit

due to contamination pellet

previously site

in the

AND

conducted

1, cross with

allowance

the oxaloacetate-

subunit

of the

inhibitor-

by grants

from

the United

enzyme complex.

ACKNOWLEDGMENTS--This States

Public

Health

Service

work

was supported

(GM-16767

and HL-12576).

References 1.

2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

King, T. E., Advances in Enzymology, 8, 155 (1966). Ohnishi, T., Winter, D. B., Lim, J., and King, T. E., Biochem. Biophys. Res. Com&n., 12, 231 (1973). Davis, K. A., and Hatefi, Y., Biochemistry lo, 2509 (1971). Vinogradov, A. D., Winter, D. B., and King, T. E., Biochem. Biophys. Res. Commun., 9, 441 (1972). Weber, K., and Osborn, M., J. Biol. Chem., 244, 4409 (1969). Kearney, E. B., Ackrell, B. A. C., and Mayr, M., Biochem. Biophys. Res. Commun., 49, 1115 (1972). Priegnitz, A., Brzhevskaya, 0. N., Wojtczak, L., Biochem. Biophys. Res. Commun., 5J, 1034 (1973). Kappen, L. S., Brodie, J. D., and Nicholls, P., Biochem. Sot. Trans. 1, 877 (1973). DerVartanian, D., Dissertation, University of Amsterdam, 1964. Keilin, D., and King, T. E., Proc. Roy. Sot., Ser. m, 163 (1960). Ryan, C. A., and King, T. E., Biochim. Biophys. Acta, 62, 269 (1962).

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