An NADH dependent cleavage of DTNB by the α-ketoglutarate dehydrogenase complex

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 32, No. 3, 1968

AN NADH DEPENDENT CLEAVAGE OF DTNB BY THE a-KETOGLUTARATE

DEHYDROGENASE COMPLEX1

J. D. Erfle Animal

Research

Research

Institute,

Ottawa

Received

Branch,

Canada Department

3, Ontario,

of Agriculture,

Canada

July 10, 1968

In the complex

titration

of sulfhydryl

by the DTNB2 method

contribution lipoic

and F. Sauer

to the total

acid.

groups

of Ellman sulfhydryl

The mechanism

in

(1959) groups

+ LipS2-E2

Reduced

-

+ E3-FAD

that

what

enzyme bound

NADH would

reduce

all

CRCH(OH)-TPP!-~1

+ co2

(1)

-f

[RCO-S-LipSHI-E2

+ TPP-El

(2)

[Lip(SH)2]-E2

2

E3-FAD + NAD+ i!

RCOCOOH + NAD+ + CoASH

to determine

+

+ CoASH 5!

[Lip(SH)2]-E2

was of interest

predicted

RCOCOOH + TPP-El

[RCO-S-LipSH]-E2

it

dehydrogenase

was made by reduced,

of the reaction

[RCH(OH)-TPP]-~~

the a-ketoglutarate

+

LipS2-E2

+ R-CO-S-CoA + reduced

(3)

E3FAD

(4)

E3FAD + NADH + H+ RCO-S-CoA

(5)

--

+ CO2 + NADH + H+

(6)

R = HOOC(CH2)2 the lipoic

sulfhydryl

and these

should

in the complex metric.

then

by reversal

be titratable.

in the presence

The nature

' Contribution

groups

of this

No. 317 from

of reactions The reaction

of NADH proved reaction

the Animal

4 and 5 (Sanadi of DTNB with

to be catalytic

has been studied

Research

et al.

sulfhydryl rather

and evidence

than

1952) groups

stoichio

is presented

Institute.

DTNB, 5,5'-dithio-bis(2-nitrobenzoic acid); TNB, 5-thio, 2 Abbreviations: 2-nitrobenzoic acid; TPP, thiamine pyrophosphate; Lips2 and Lip(SH)2, FAD, flavin adenine dinucleotide. oxidized and reduced lipoate (or lipoamide); E2 is dihydrolipoyl transuccinylase and El is a-ketoglutarate decarboxylase; E3 is lipoamide dehydrogenase (E.C. 1.6.4.3).

562

to

Vol. 32, No. 3, 1968

BIOCHEMICAL

demonstrate

of DTNB with

the reaction

succinylat:ion is

during

reduced

by lipoamide

the internal this

catalysis

is

the

of the

of lipoic

acid

taining

0.5

density

centrifugation

overall

G-200

and elution

Traces

study

additions

DTNB, 1 umole;

given

dehydrogenase

complex

optima

and 9.2.

of the reaction

of

by

was to replace

the

on a 2.5 x 40 cm (pH 7.0)

removed

(1966).

con-

by sucrose

The enzyme prepara-

2 to 4 units

per mg in

the

Boehringer

Biochemical

Mannheim

from

Corp.

N-ethyl

Corporation.

the increase potassium

NADH and iodoacetamide

in absorbancy

phosphate,

at 412 mu in a

(pH 7.0),

dehydrogenase,

5 ug;

of preincubation

50 umoles;

and purified

and the

order

CL-

of

and tables.

and Discussion

catalytic

illustrated The Km for

was 1 umole

dehydrogenase

Co., from

lipoamide

figure

of the is

from

Chemical

The details

in the

The NADH dependence

Lipoamide

of

heart

phosphate

were

--et al.

activities

Results -

metry

to that

from bovine

M potassium

by Ishikawa

dehydrogenase

contained

dehydrogenase.

at 7.6

formed

The significance

chromatography

dehydrogenase

of assay was to follow

umoles;

are

4).

modification

with

0.05

of pyruvate

of Nutritional

Incubations

ketoglutarate

thus

fashion

was isolated

The only

with

from Aldrich

was a product

NADH, 0.15

undergoes

6).

Sigma Chem. Co. and lipoamide

1 cm cuvette.

complex

had specific

(reaction

DTNB was purchased

The method

The dithiol

of reaction

chromatography

as described

this

reaction

maleimide

which

and Methods

(1967).

gel

mM EDTA.

in

--et al.

phosphate

of Sephadex

used

reaction.

(reverse

dehydrogenase

of Hirashima

cellulose-calcium

tions

sulfhydryl

discussed.

The o-ketoglutarate

column

acid

and NADH in an analogous

Materials

the method

lipoic

overall

dehydrogenase

dithiol

react-ion

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

alone

cleavage

in Figure

of DTNB by the a-ketoglutarate

1 (curve

DTNB was 1 mM at both of NADH oxidized was unable

563

1).

This

pH values.

per mole

to catalyze

reaction

has pH

The stoichio-

of DTNB reduced.

this

reaction

(Figure

1,

Vol. 32, No. 3, 1968

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TIME (MN)

cleavage of DTNB by the a-ketoglutarate dehydrogenase Figure 1. The enzymatic complex, The assay system is described in the Methods. Curve 1 - a-ketoglutarate dehydrogenase, 53 pg, (SQ. Act. 4 units/mg); curve 2 - same as 1 but preincubated 2 min. with NADH and succinyl-CoA (0.098 umoles/ml); curve 3 - same as 1 but preincubated 2 min. with a-ketoglutarate (0.1 umolefml); curve 4 - lipoamide dehydroAt the point indicated by the arrow the reaction was started by genase (25 ug). the addition of NADH (curves 1 and 4) or DTNB (curves 2 and 3). Table

I.

Properties cleavage

of the a-ketoglutarate of DTNB. Other

Enzyme additions*

dehydrogenase

additions

catalyzed

Activity

a-KGDH (native)

none

100

Lip.DH

none

0

Lip.DH o-KGDH (denatured)**

lipoamide + Lip.DH

(0.01 none

umole)

780 78

o-KGDH (denatured)

+ Lip.DH

Succinyl-CoA

78

a-KGDH (denatured)

+ Lip.DH

a-ketoglutarate

100

The reaction was run at pH 7.0, * The complete system is described in Methods. dehydrogenase complex. and initiated with DTNB. a-KGDH is a-ketoglutarate Lip.DH is lipoamide dehydrogenase. ** a-KGDH was denatured by a 10 min. incubation in 2.5 N KOH of 30°. paration was neutralized to pH 7.0 and buffered before assaying. 564

The pre-

Vol. 32, No. 3, 1968

curve

4),

unless

requirement

for

resides

with

8lOCHEMlCAL

catalytic

succinylase).

protein That

promotes

dehydrogenase

complex.

The participation in DTNB cleavage results

from

succinyl-CoA in the

tional

bound

evidence

Table

II.

the

case is

NADH.

demonstrated

moieties

demonstrated

results

was inhibitory the role

of lipoic

I where

complex

acid

tran-

lipoamide

2 and 3) by the inhibition

with

a-ketoglutarate of the

or lipoic

acid

and by reversal

Neither

cleavage

probably

transuccinylase

a-ketoglutarate

was denatured

in the

the

a-ketoglutarate

succinylation

and NADH.

Thus

the dihydrolipoyl

the case of a-ketoglutarate

when the

I). complex

of denatured

1, curves

in enzymatic

1 and 2 in

in

in Table

of the enzyme complex

This

(Table

of dihydrolipoyl

(Figure

the case of succinyl-CoA

for

(i.e.

acid

lipoic

(Table of DTNB is

I).

Addi-

provided

by

Effect of NADH on NEM and iodoacetamide inhibition of DTNB cleavage by the a-ketoglutarate dehydrogenase complex *

conditions:

NADH --

Iodoacetamide

+

residues

of the

Preincubation

+

acid

added

dehydrogenase

in the presence

further

3-5 in

were

DTNB cleavage

enzyme by reactions

succinyl-CoA

lipoic

preincubation

plus

of reactions nor

is

of lipoamide

of the a-ketoglutarate

such is

dehydrogenase

which

quantities

the remainder

its

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

NEM

Enzyme activity** G> 100

+

95

+

0 +

78

+

0

* Preincubations contained in 1 ml: pmoles; NADH, 1.2 umoles; NEM, 1 A 10 min. preincubation of enzyme after the addition of sulfhydryl of the last four incubations was to titrate sulfhydryl reagents. ** The enzyme preparations were hydrogenase (5 pg) was added addition of NADH.

Tris-Cl (pH 7.0), 50 umoles; EDTA, 0.2 pmole; iodoacetamide, 2 umoles; oKGDH, 3.6 mg. and NADH was followed by a 15 min. incubation reagents. After the incubation a 0.25 ml aliquot mixed with a stoichiometric amount of cysteine

assayed as described in Methods. Lipoamide The reactions were started to each assay.

565

deby the

Vol. 32, No. 3, 1968

the

inhibition

mide

(Table

studies II).

the reversal

by both

cleaves

These

complex

mechanism

bond

dependent also

is

and iodoaceta-

on NADH and hence

show that

oxidized

lipoic

the native acid

shown as one in which

between

6 of lipoic

Lipoamide E2-NHCO(CH2)4-CH

is

only

of DTNB cleavage

at carbon

N-ethylmaleimide

results

contains

the disulfide

sulfhydryl

reagents

compounds

4 and 5.

dehydrogenase

dehydrogenase

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the sulfhydryl

Inhibition

The proposed

the

with

of reactions

ketoglutarate

Since

BIOCHEMICAL

acid

lipoic is

acid

the

on o-

residues. lipoamide

and thionitrobenzoate.

one reported

to undergo

dehydrogenase

- CH2 - CH2 1 S

:

+q

E2-NHCO(CH2)4-jH

- CH2 - iH2 SH

NAD+

SH

TNB + NAD+

succinylation reaction.

(Gunsalus This

is

et al.,

consistent

1958) with

it

is depicted

inhibition

caused

as the one involved by conditions

in

the

favouring

succinylation.

Conclusions The results acid-TNB

disulfide

to succinylation

presented

demonstrate

by lipoamide have

led

the NADH dependent

dehydrogenase.

to the proposed

Inhibition

mechanism.

566

cleavage

of the

by conditions This

model

reaction

lipoic conducive has been

Vol. 32, No. 3, 1968

used to study all

for other This

is

this

caution

chain

as well.

demonstrating

the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of lipoic acyl-CoA

of inhibition

acid

esters

moieties

in

and may have

The reaction

catalytic

involvement

cleavage

of a dithiol

the inhibition application

in studies

may in addition of protein

of the over-

provide

bound

of

a useful

lipoic

acid

enzymes. enzyme-catalyzed

to the best

that

the

by long

types

tool in

the involvement

reaction

other

BIOCHEMICAL

of our knowledge

reaction

is

of a more general

to investigators

presence

a hitherto

who attempt

formed

unreported

reaction

reaction.

or non-specific the titration

from

with

DTNB

The possibility

nature

should

serve

of enzyme sulfhydryl

as a

groups

in

of substrate.

References Ellman, G. L., (1959), Archives Biochem. Biophys., 82, 70. Gunsalus, I. C., and Smith, R. A., (1958), in Proceedings of Symposium on Enzyme Chemistry, p. 77. Maruzen, Tokyo, Hirashima, M., Hayakawa, T., and Koike, M., (1967), J. Biol. Ishikawa, E., Oliver, R. M., and Reed, L. J., (1966), Proc. 2, 534. Sanadi, D. K., Littlefield, J. W., and Bock, R. M., (1952),

567

the

International

Chem. a, Natl. Acad. J. Biol.

902. Sci.

(U.S.)

Chem. 197,

851.