A study of dithiothreitol inactivation of the enzyme rhodanese

Vol.67,

No.1,1975

BIOCHEMICAL

A Study

AND BIOPHYSICAL

of Dithiothreitol

RESEARCH COMMUNICATIONS

Inactivation

of

the

Enzyme Rhodanese Seung Department Hanover,

Received

September

K. Kim and Paul M. Horowitz of Chemistry, Dartmouth College New Hampshire, 03755, USA

16,1975 Summary

When air oxidized, partially inactivated rhodanese (EC 2.8.1.1) is treated with dithiothreitol (DTT) to regenerate the reduced essential sulfhydryl group there is an initial reactivation followed by an anomalous slower inactivation. Fully active enzyme shows only inactivation. The inactivated enzyme may be completely reactivated on long incubation with the substrate thiosulfate ion. None of the normal potentialities of DTT appear to be The results are interpreted in terms of responsible for the inactivation. disulfide formation between DTT and an essential enzymic sulfhydryl group with the resulting complex being stabilized by secondary interactions which are particularly favorable due to similarities between DTT and lipoic acid--a normal sulfur acceptor substrate.

Introduction The enzyme sulfate

using

sulfide,

reduced

and oxidized

reduced loses

sulfhydryl activity

tial

rhodanese

sulfhydryl

(DTT)l

groups

(3)

fide

cleavage:

oxidize

critical

chelate

metal

1

abbreviations:

reduction

acceptor

lipoic

acid

(1).

active

enzyme

that

purified

Fully

is often

on storage

due at

least

groups

to disulfides

is found

the

enzyme

residues

and is enzyme

systems

Autooxidation

of (6)

partially

rhodanese

disulfides that

found

DTT, dithiothreitol.

and produce

to the

is

in at

is

thio-

sulfite,

contains

only

rhodanese

oxidation

attempted

innocuous rapidly least

DTT produces

and DTT by virtue

(7).

of

of essen-

(2).

of oxidized

reduces

ions

the

as a sulfur

and it

DTT can affect

catalyze

acid

groups

which it

will

lipoic

When reactivation to1

(EC 2.8.1.1)

with

with free

inactivated two ways distinct

hydrogen of

its

peroxide dithiol

dithiothreisulfhydryl (4,5). from which structure

disulcan can

Vol.67,No.l,

1975

These

results

of rhodanese metal

in the

We report

the

Procedure

Crystalline Horowitz

were

the

For

were the

were

tion 32,600 zero

the

kept

at

was used

1 shows

by a slower

rate

inactivation. buffer

time

ratio

activity

raising

to

was used

then

(0.74

solution

with

DTT were

of

the

activity

of

rhodanese

inactivated

enzyme

sample

(0.51

IU/pg)

all

DTT:enzyme,

there

essentially

complete

ratio

5 shows that the

at enzyme

3 shows

Curve

added

original

Curve

is

weight

the

DTT.

9:1,

concentra-

M DTT were

enzyme

and

IU/ug)

a molecular

Both

the

added

an enzyme

of 0.1

solution.

through

experiment.

rhodanese

and assuming

(11).

of time.

course

DTT:enzyme

for

the

(10).

procedure

were

at O"togive

and the

as obtained.

was bubbled

throughout

enzyme

other

used

biuret

of enzyme

quantities

of

of Wang and Volini

nitrogen

protein

control

and were

without

which the

grades

All

to controls

of

inactivation

that

2 This

the

partially

4 shows

of

method

Calbiochem.

a modified

pH 7.g2

as a function

DTT relative

DTT at a molar

of

as the

0°C and assayed

from

crystalline

on biuret

which

oxidized,

by the

nitrogen

buffer

prepared

air

susceptibility importance

was prepared

using

experiments

1 ml of the

added

reported

inactivation.

quantities

with

to

with

with

blanketed

Microliter

Figure

the

observed

deaeration,

enzyme).

time

solution

measured

in 0.2 fi Tris

for

the

by the method

Microliter

3 x 10 -5 E (based

of

of

DTT on rhodanese

commercial

requiring

inactivation

was dissolved

of

DTT was obtained

20 minutes.

solutions

for

was measured

For experiments for

and the

effect

rhodanese

available

concentrations

buffers

in view

(8).

the

liver

best

Enzyme activity Protein

RESEARCH COMMUNICATIONS

and Results

bovine

the

of

mechanism

and DeToma (9).

materials

(2)

mechanism

studies

to elucidate

interesting

inactivation

catalytic here

Experimental

AND BIOPHYSICAL

are especially

to oxidative

ions

attempt

BIOCHEMICAL

studies

434

to 2OO:l fully

is

presented

that is

followed

by 70 minutes. increases

rhodanese here.

when an

treated

a reactivation

greatly

active

samples

(0.73

Curve the IU/ug)

of

Vol. 67, No. 1, 1975

Figure

1.

BIOCHEMICAL

Time

course

Curve

of rhodanese

= 0.51

danese

= 2OO:l;

Curve B-mercaptoethanol degree

of

pared

with

addition there

at

that

3 for

a 10 fold tion bility

of

molar

that

and indicates Trotta,

Figure excess

enzyme

of

molar

same molar

over

only

excess

rapid

gives This

of DTT. addition

activity

It

DTT:rho-

details.

that

enzyme

specific

specific

experimental

incubation.

before

enzyme

= 200:1,

for

B-mercapto-

3 with

of comparison

an 80 minute

2,

= 9:1,

same as curve

DTT displays

excess

1 shows the of

DTT over

inactivation. the

only curve

monothiol a small can be com-

may be noted

to the

enzyme

in

sample,

at any dithiothreitol:enzyme

that

results

an oxidation al.

have

indicated

effect enzyme This

was apparent.

inactivation

et

4,

Curve

ratio

1OO:l.

1 of

the

the

on the

up to and including Curve

excess

dithiothreitol.

DTT:rhodanese

See text

when DTT was oxidized

was no effect

3,

1 shows as a point

during

with

5, DTT:rhodanese

IU/ug.

a 9 fold

inactivation curve

Curve

molar

2 of Figure

Curve

RESEARCH COMMUNICATIONS

deaerated;

Curve

IU/ug,;

= 0.73

a 200 fold

= lO:l,

= 9:l;

activity

activity

with

inactivation

1, DTT:rhodanese

ethanol:rhodanese

treated

AND BIOPHYSICAL

from is

when the added.

result the

system In this

appears

chelation

was deaerated case

and

no inactiva-

to eliminate

the

of an essential

possi-

metal

required. that

435

low

concentrations

of

DTT can pro-

ion

Vol.67,

No. 1, 1975

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

0.8 -

0

Figure

2.

Time

20

course

danese.

of

40 TIME

thiosulfate

Rhodanese

of control;

Curve

of

duce

hydrogen

0.5

in the

peroxide

susceptible

enzymes.

centrations

(6).

0.71

IU/ug)

in 0.2

the

enzyme

hours

of

with

In the

to

Incubation

20 rn! DTT rather

from

those

the

complete

in the tivating

than

of Trotta,

2 shows reactivated,

presence enzyme

ten

of that

of

added

active

stabilizing

enzyme relative

of 62.5

mp2 sodium

relative

to the

that to

hydrogen

resulted

last

observed

has been

is

436

mg/ml,

DTT con-

(0.85

mg/ml, of

after 0.71

differ

peroxide

two Ill/us) loss

markedly

production

as

here.

by long

shown

high

in complete

inactivated

thiosulfate. control

peroxide

results

hydrogen

a control,

inactivate

30% inactivation

enzyme

These

phenomena

details.

(0.35

and eliminate the

experimental

enzyme

the

minutes.

et al.

explanation

Figure completely

than

without

curves density

by using

than

to

to an optical

of rhodanese less

relative For

can irreversibly

incubation

rho1, activity

sample

for

be abolished

gave

Curve

sample.

See text

peroxide

a control

reactivating

which

case

mg/ml.

1 corresponds

could

present

DTT inactivated

reactivating

assay.

effect

rn! hydrogen

in less

of

on autooxidation

incubation.

of activity

of

value

standard

This

relative

of

3, activity

of

= 0.91

2, activity

1 and 3 an ordinate

80

reactivation

concentration

Curve

control;

60 (hrs.)

by DTT can be incubation

at 4'C

The activity

of the

reac-

by curve

2 and begins

at

Vol.67,No.

zero

1, 1975

relative

hours.

activity

Figure

(curve these

BIOCHEMICAL

two curves

substrate

the

the

individual

time (curve

of

thiosulfate

inactivation

in the

was 4OO:l

control

courses

values

for

the

An ordinate

in the

assay

or greater,

mixture. there

at 90

control value

of

1 for

system.

by DTT may be slowed

incubation

(DTT:enzyme

RESEARCH COMMUNICATIONS

the

3).

to an OD = 0.50

rate

incubation

to 96% of

sample

corresponds

of thiosulfate:DTT 90 minute

shows

reactivating

In addition the

at t=O and rises

2 also

1) and the

AND BIOPHYSICAL

by including

When the

molar

was no inactivation

ratio during

a

enzyme

acti-

= 8:l).

Discussion In the vitythis

past

indicated

potent

in the

appears

responsible

for

for

but

The results

is realized

the

that the

been assumed

involvement

reductant

It

model

has often the

disulfide

gen peroxide. tials

it

may also

metal

indicate

of

rhodanese.

interaction

DTT is ions

that

between

inactivation.

we have

Ku

chelate

bond.

here

inactivation

a direct

effects

when DTT affected

of a disulfide

presented

observed

that

a

and produce

none of

DTT and the

Presented

in fact

below

is

these

enzyme

hydropoten-

is

a mnemonic

described:

I

II

ESH

ES-SE

HS, I

I

D

HS'

ES-S

HS, D

HS'

ESH + ES-,S

HS'Dw

s sv:o ESH •t D:t S

Scheme essential

II shows rhodanese sulfhydryl

species

may interact

sulfhydryl

group

groups with interchange,

inactivated from

by the separate

a molecule

oxidation

active

to

sites

(2).

of DTT to participate

releasing

one active

4.17

a disulfide

enzyme

This

of

two

inactive

in a disulfideunit

and producing

Vol. 67, No. 1, 1975

BIOCHEMICAL

an enzyme-DTT

complex.

would

cyclize

rapidly

form

of DTT thus

zation for

of

the

binding

forms last

in this

group

disulfide

formation

tion.

is

is

is

with

require

Scheme would

the

is

suggested

Under

II).

cannot

This

be observed

Therefore,

its

the enzyme

of

thio-

necessity

free

active

conditions directly,

occur

sequence

for site

there

which

readily

in this

the

and the

oxidizing complex

that

(12).

with

DTT-enzyme

dithiol

absence

oxidation

rhodanese

stabili-

responsible

at which

in the

ring

that

(1).

rate

DTT

oxidized

aliphatic

enzyme

any net

top.

the

stable

rhodanese

the

Normally

the

1,3

that

Active

I.

step.

interactions

acid--a

interesting

at the

to give

inactivation

secondary

inactivate

above

(cf.

the lipoic

also

shown

It

shown as limiting

in Scheme

be stabilized Only

will

to produce

complex

It

steps

suggested

sulfhydryl

would

using

reactivated. acid

step

reduced

scheme

None of the is

occurs,

RESEARCH COMMUNICATIONS

a reactivation

reactivation.

significant

lipoic

oxygen

last the

substrate

a kinetically

sulfate,

in the

complex

may be fully

represents

completing

the

step

This

AND BIOPHYSICAL

can be

again

on deaera-

in the

presence

of oxygen.

Acknowledgment Acknowledgment administered

is made to the

by the

American

donors

Chemical

of The Petroleum

Society,

for

Research

support

of this

Fund, research.

References 1.

Volini,

M.,

2.

Wang,

3.

Cleland,

4.

Kim,

5.

Man, M.,

S-F.,

S.K.,

and Westley, and Volini,

W.W. (1964)

J.

M. (1968)

J. J.

Biochemistry,

and Horowitz, and Bryant,

(1966)

R.G.

Biol. Biol.

Ckem., Chem.,

241,

5168-5176.

243,

5465-5470.

3, 480-482.

P. (1974) (1974)

438

Fed. J.

Biol.

Proc., Chem.,

33,

1379. 249,

1109-1112.

BIOCHEMICAL

Vol. 67, No. 1, 1975

6.

Trotta,

P.P.,

Pinkus,

L.M.,

AND BIOPHYSICAL

and Meister,

A.

RESEARCH COMMUNICATIONS

(1974)

J. Biol.

Chem.,

249, 1915-1921. 7.

Gracy,

R.W.,

8.

Volini, Proc.,

M., Van Sweringen, 34, 495.

9.

Horowitz,

10.

Wang,

11.

Zamenhof,

12.

Volini,

P.,

S-F.,

and Noltmann,

E.A. B.,

and DeToma, and Volini,

DeToma,

Wang,

F. (1970)

F.,

Enzymol.,

and Westley,

*

J. Biol.

439

Chem.,

S-F.,

and Leung,

J. Biol.

Chem.,

J. Biol.

M. (1973)

S. (1957) Methods M.,

(1968)

Chem.,

243,

4109-4116.

J. (1975) Fed. 245,

248,

984-985. 7376-7385.

3, 696-704.

J. (1967) 242, 5220-5225.