Multiple interactions of threonine with an aspartokinase-homoserine dehydrogenase complex

Vol. 30, No. 2, 1968

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MULTIPLE

INTERACTIONS

OF THREONINE WITH AN ASPARTOKINASE-

HOMOSERINE DEHYDROGENASE COMPLEX Glenn

Cunningham,

N.

From the

Clayton

Department

Stephen

Foundation

of Chemistry,

B. Maul;

and William

Biochemical

The University

Shive

Institute

and the

of Texas,

Austin,

Texas

78712

Received December 26, 1967

It

has been proposed

aspartokinase

and homoserine

Escherichia during

coli

K-12

multiple

and control

of both mutation

lacking

homoserine

the absence

as a result

at higher of these greater

*

to a lower

to cause

first,

concentrations

than

that

NIH Predoctoral

together by threonine

was modified

aspartokinase

from

was found

to dissociate

weight

unit

the effects

dehydrogenase

supplementation

a

by

mutant

with

of E. &

molecular

weight

9723

in the

loss

needed

for

reversible

association

159

(3).

Threonine

50

to

formed has

association to inhibit

of the complex.

concentrations

Fellow

is

form and then

threonine

upon

which

a reversible

each of the activities requires

of threonine

complex

at low concentrations,

a higher

into

activities

stabilized

inhibition

molecular

concerns

and homoserine

of the complex

activity

of

are fractionated

and are

the

in one protein

(2).

of lysine

found

activities

Furthermore,

investigation

an aspartokinase

reside

by threonine

dehydrogenase

control

The present

both

activities (1).

of threonine-sensitive

dehydrogenase

of purification

of threonine

of threonine

the activities

because

stages

a single

been

that

100

to a higher

Inhibition times

molecular

Vol. 30, No. 2, 1968

weight

BIOCHEMICAL

form.

In addition

of the two activities actions

&erimental

until

hours

(4)

0.03

the

phosphate

dry

After

150,000

x g, the

this

fraction

potassium

The denatured

into

protein

was then

stored

at 4" C.

as will

was recovered

cell

0.03

and (20 to 30

one hour

at

from

up in 0.02

g

g @mercaptoethanol

and heated

for

by centrifugation,

to use,

of 50%. This

the protein

and dissolved

i 8-mercaptoethanol

5 minutes and ammonium

a concentration

prior

at

The protein

0.03

to give

5

by precipitation

and taken

containing

Just

in 0.02

buffer

for

+ 10

oscillation

extract

by centrifugation

phosphate,

from

in buffer

plus

additives

be indicated). Gel filtration

of Andrews

with Sucrose

(5).

by the method

of Martin

the above described

a Sephadex density

G-100 was performed

gradient

and Ames (6)

buffers)

Model

L-2 with

using

an ISCO Density-Gradient

out

by sonic

10 ml fractions

fraction

(0.5

twice

in fresh

disruption

was removed

in salts-

2 mM -- L-threonine

was fractionated

supernatant

_M potassium

containing

liquid

to the

(0.02

washed

crude

was added

fraction

inter-

at 660 rnp (about

of the

sulfate

this

- 0.22

harvested,

(pli 6.8)

divided

multiple

and &-methionine

by centrifugation

buffer

inhibition

cultured

35 and 48X saturation.

was recovered

and 5 rn_ threonine,

9723 were

resuspended

for

between

phosphate

at 55".

per ml)

supernatant

ammonium sulfate

were

(pH 6.8)

centrifugation

with

suggests

&-lysine

was 0.18

and then

weight

10 kc.

with

Cells

E 8-mercaptoethanol,

mg of cell

of 5. coli

density

buffer

which

of the

enzyme complex.

supplemented

at 30").

in the nature

observed

--Cells

the optical

incubation

potassium

with Methods.

medium

each),

differences

have been

of threonine

glucose

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

a 65 K rotor.

for

with

centrifugation a 5-207.

210 minutes

Samples

from

Fractionator.

by the method

sucrose

at 65,000 the

was performed (in

rpm in a Beckman

gradient All

gradient

were

procedures

collected were

carried

at 4" C. Aspartokinase

was assayed

as described

160

by Lee --et al.

(7).

liomoserine

Vol. 30, No. 2, 1968

dehydrogenase absorption

BlOCHEMlCAL

was assayed

by measuring

at 340 rnp for

(pH 8.8)

containing

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

a period

100 @ Tris,

reduction

of NADP+ by changes

of 1.5 minutes

in

in a 1.0 ml mixture

80 mFJKCL, 50 mFJ&-homoserine

and 0.4 mFJ

NADP+. Results

and Discussion.

concentrations

of threonine

and homoserine

dehydrogenase

association Figure

to form

upon

a higher

1, the retention

considerably

--The

pattern

molecular

weight

threonine

the

range

complex molecular

from ;.

coli -.-

weight

of 80,000 is

mlbvithout

with

- 100,000

complex. G-100

a reversible

is decreased eluting

buffer.

corresponds

whereas

Effluent,

lowest

As shown in

in the

not appreciably

L-Threonine)

9723 is

of threonine

the

aspartokinase

on Sephadex

60

Effluent,

effect

of 20 pI4 threonine

in the absence

enzyme complex

observed

the threonine-sensitive

of the complex

in the presence

The retention

first

in the presence

retarded

68

76

mlkith

to the

by the

84

of gel.

92

L-Threonine)

pattern of threonine sensitive aspartokinase and FIG. 1. Elution homoserine dehydrogenase complex on Sephadex G-100 (2.0 x 60 cm flow rate 20-25 ml/hr) in the absence of and in the presence of threonine 20 @. A - Threonine sensitive aspartokinase activity, B - Threonine sensitive homoserine dehydrogenase activity, C - Threonine sensitive homoserine dehydrogenase activity in the presence of the substrates of the aspartokinase (aspartic acid, 10 or& and ATP, 10 m?J with Mg*, 1.6 a&i). All columns were run in 0.02 g PO4 buffer containing 0.03 z @-mercaptoethanol plus additives as indicated. Aspartokinase activity is expressed as micromoles of B-aspartylhydroxamate produced per ml during a 20 minute incubation. Homoserine dehydrogenase activity is expressed as millimicromoles of NADPH produced per ml per minute.

161

Vol. 30, No. 2, 1968

BIOCHEMICAL

The mobilities to that the

of the enzyme

of well

characterized

sedimentation

coefficient

of the A2gO of the sucrose ponded for

to the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

complex enzymes

in order

(S20,w)

under

gradient

of the

activity.

This

thus

indicating

an apparent

remotely

possible

gradient

were

to obtain

a small

enzyme complex

compared

an estimate

each condition.

revealed

position

enzymatic

in a sucrose

of

Tracings

peak which

as determined

corres-

by analysis

gave S20 w values of 6.3 without , threonine and 9.7 with 20 CIM_threonine. These S20 w values correspond , to an approximate molecular weight of 100,000 - 120,000 and 195,000 220,000, it

is

complex

could

above.

The reversibility

of threonine

from

appearance G-100

result

of the

forms

lower

indicates cular

weight

the form

an appreciable

the

association

dehydrogenase the type

dehydrogenase

weight

form which

form

as a result

at considerably reaction

higher

(Fig. curve

the 2);

Either

The data

each activity. is

with unit

consistent

162

its

for

as the higher

resulting

there

mole-

alone

has

in enhanced than

of

causes activity.

necessary

for

and the homoserine is

a difference

increasing

concentrations

inhibition

of homoserine

with

1)

in conformation

substrates

aspartokinase

substrates

in Fig.

substrate

concentrations

with

by the

changes

however,

obtained

an elution

molecular

as shown

as well

Apparently

inhibits

by threonine

form

of binding

in the

by Sephadex

and lower

Mg*

activities.

dehydrogenase

resulted

dehydrogenase

and ATP with

both

by removal

was obtained.

of homoserine

weight

noted

of 2 @ threonine,

volume

although

of the enzyme

as indicated

of the higher

effect.

activities

for

that

shape

was demonstrated

weight

low molecular

activating

of inhibition

threonine

in the mobilities

acid

in the homoserine

Threonine

changes

elution

contains

aspartokinase

changes

observed

at a concentration

(aspartic

that

in the

molecular

activation

weight

changes

molecular

a broader

of aspartokinase

of molecular

drastic

between

with

doubling

of the association

Also,

Appreciable

the

in the

intermediate

weight

that

the higher

filtration.

pattern

method

independent

interaction

in of

Vol. 30, No. 2, 1968

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

L-Threonlne,

mM

L-Threonine,

mM

FIG. 2. Effect of threonine on the activity of the threonine aspartokinase and homoserine dehydrogenase. Activities expressed described in Fig. 1.

of an inhibitor

at a single

of the aspartokinase interdependence

activity

the complex

could

site

or alternatively,

inhibitor.

cause

until

could

The initial

an enhancement initial

change

be expected

in the other

complex

under

appropriate

conditions.

binding

sites

lie

points

moieties

with

tietween

points

would binding with

facilitate

preferred

site

similar

of association

binding

so that

upon its

with

little

163

effect.

inhibitory to the

initial

binding

of the changes initial

for

of

conformational associated

be expected

syseaetrical

may be essential

exerting

little

of the

could

inhibitor

at a subsequent

bound

from

portion

This

of one part

changes

one site

is also

some

of the

analogous

active

with

binding

have

resulting

conformations

of association

at more than the initial

catalytically

between

could

to facilitate

changes

the inhibition

binding

of the

binding

a subsequent

The conformational

the inhibitor

however,

to be more complex,

sites.

the

upon activity

site;

appears

of inhibitor

with

effect

regulatory

sensitive as

complex

if

these

within

in the distances inhibitor part.

effective

binding Alternatively, inhibition

Vol. 30, No. 2, 1968

Kinetic reported

(8)

BIOCHEMICAL

studies'

indicated

that

but

magnesium

ion

aspartokinase

also

activity

of the higher

addition,

the

activity

is

appears

+

of threonine

in molecular

weight

unit

kinase

due to interaction

hibition

of homoserine

however,

complex

formed

which

is

as determined

by gel

the

weight

there

formation presence

filtration.

In

dehydrogenase

and magnesium

ion.

Thus,

can each be caused and the aspartokinase

are

four

the enzyme complex:

the

to occur inhibition

and (3)

(2)

dehydrogenase. manner

inhibition

(1)

inter-

reversible with

cooperative

increase a lower

inhibition

molecular

of asparto-

at two or more sites,

and (4)

The inhibition

aspartokinase

by either

of homoserine

reversed

distinguishable

of threonine

of threonine

is

acid

in the

of the homoserine acid

of

interaction.

in a competitive

neither

in molecular

the

that

of the enzyme,

can be reversed

or prevent

due to interaction

weight

inhibition

ml$ do not cause

of threonine,

with

as previously

aspartic

of the two inhibitions

appears

acid

the threonine

manner.

by aspartic

a two stage it

aspartic

reverses

by threonine

concentrations

to involve

only

However,

or 0.4 m$

reversed

In summary, actions

weight

and either

at different

(3.3

inhibition

not

association

ion

molecular (0.02

not

in a competitive

(10 mb$ and magnesium

of threonine

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of the Mg

+t or aspartic

dehydrogenase nor +i acid or Mg .

by aspartic

in-

acid; the

increase

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Patte, Acta,

J. C., Truffa-Bachi, 128, 426 (1966).

2.

Janin, J., Truffa-Bachi, Conmun., 26, 429 (1967).

3.

Lansford, E. M., Jr., Lee, N. M., Res. Connnun., 25, 468 (1966).

and Shive,

4.

Anderson,

Sci.,

E. H.,

1 E. M. Lansford,

Jr.,

Proc.

P., P.,

Nat.

and Cohen,

and Cohen,

Acad.

G. N.,

32,

N. M. Lee and W. Shive,

164

G. N.,

Biochim.

Biochem.

Biophys. Biophys.

W., Biochem.

Biophys.

120 (1946).

Unpublished

data.

Res.

Vol. 30, No. 2, 1968

P.,

BIOCHEMICAL

Biochem.

J.,

91,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

5.

Andrews,

6.

Martin,

7.

Lee, Res.

8.

Stadtman, E. R., Cohen. G. N., LeBras, H ., J. Biol. Chem., 236, 2033 (1961).

R. G. and Ames, B. N., N. M., Lansford, E. M., Commun., 26, 315 (1966).

222 (1964). J. Biol. Jr.,

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IRK

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W., Biochem.

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