Dissociation properties of aspartokinase I-homoserine dehydrogenase I extracted from a missense mutant of E. coli K12

Vol. 60, No. 1, 1974

BIOCHEMICAL

DISSOCIATION

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

PROPERTIES OF ASPARTOKINASE I-HOMOSERINE

DEHY~RO~ENAS~ I EXTRACTED FROM A M~SSEN~E MUTANT OF E.coli Jacques Service Institut Received

July

15,

K12

THEZE and Michel

de Biochimie Pasteur, 75015

VERON

Cellulaire, Paris, France.

1974

The threonine sensitive aspartokinase-homoserine SUMMARY. dehydrogenase devoid of aspartokinase activity has been extracted from a missense mutant of E. coli K12 and some of its properties have been investigated. The genetic localization of the corresponding mutation indicated that the amino acid replacement lies in the kinase segion of the molecule. The cooperativity of threonine inhibition of the homoserine dehydrogenase activity is lowered. The measurement of the molecular weight of the enzyme in presence or absence of threonine indicates that the molecule dissociates more easily than the wild type enzyme. These results are discussed in view of the recent structural model proposed for aspartokinase I-homoaerine dehydrogenase I. Aspartokinase

synthesis, (1).

is

Each

that

of

w,

ties

the

which

the

structural I is

In

composed

this

homoserine 0 1974

chain

(2).

of

A model

aspartokinase

devoid

of

219

the

the

and me

coding I activi-

proposed

role

for

I (4) in

the in

the

or-

enzyme.

threonine

aspartokinase

shown

I-homoserine

dehydrogenase a central

= 86000)

has been

mL

recently

bio-

independent

dehydrogenase

of

we describe

it

and

two

aspartokinase

homoserine

play

the

by two

cistrons

structure

by Academic Press, Inc. in any form reserved.

for

has been

globules

tetrameric

dehydrogenase

two

threonine

for

Moreover

coding

2.7.2.4.

(M.W.

sites

be carried

I-homoserine

communication,

of reproduction

to

in

subunits

catalytic

shown

I and the (3).

the

the

I (E.C.

involved

identical

gene

aspartokinase of

four

been

polypeptide

of the

of

the

respectively

ganization

Copyright AI1 rights

have

aspartokinase

structure

enzyme

possesses

which

dehydrogenase for

composed

subunit

activities regions

dehydrogenase

a bifunctional

1.1.1.3),

E.C.

I-homoserine

activity

sensitive extrac-

Vol. 60, No. ?,1974

ted. from mutant

BIOCH~CAL

strain

Gif

and some of

I.O~MI.. its

AND BIOPHYSICAL

The genetic

dissociation

RESEARCH CO~~NI~TIONS

characterization

properties

are

of

the

presented.

MATERIALS AND METHODS

Bacterial

strains.

Gif

106Ml

homoserine of III

has been

and

diaminopimelic

aspartobinase (5).

Buffers

II

Strain

Biochimie

is

from

and measurement extracts

phosphate

of

were

pH 7.2,

chloride also

acid

homoserine

HfrH

selected auxotrophy

dehydrogenase the

in

basis

of

a strain

II

collection

devoid

and aspartokinase

of the

Service

de

activity.

prepared

in

2 mM magnesium

contained

dehydrogenase

physiological

direction

as substrates

(6).

hydroxamate strain

150 m!4 potassium

using

this

buffer

AT).

activity

was measured

NADPH and aspartate

Aspartokinase

test

A : 20 mM potassium

When indicated,

2 mM L-threonine(buffer

Homoserine

of

buffer

titriplex,

and 1 mM dithiothreitol.

Growth

on the

Cellulaire.

Crude

the

previously

activity

in

the

semialdehyde

was estimated

using

(7).

HfrH

and Gif

l.06Ml

and pressration

of

the

crude

extracts. Overnight in the

the

same medium

bacteria

oscillation below).

were in

For

grown

cultures, to

strain

After

108 bacteria/ml.

harvested,

buffer

on minimal

washed

106 Ml,

the

three

and disrupted

A or AT depending Gif

medium

on the

medium

were

diluted

generations, by sonic

experiment

was supplemented

(see with

-3 -3 M, D,L-homoserine D,L-isoleucine 2 x 10 M, D,L-arginine 2 x 10 -4 3 x 10 M, L-lyeine lLO-3MMand -diaminopimelic acid 10 -4M .

220

BIOCHEMICAL

Vol. 60, No. 1, 1974

Molecular

weight

with

previously

the

(M.W.

heart

with

= 12,000).

In

buffer.

the

added

as an ihternal

proteins

alcohol

experiment

alcohol

room

had been : beef

dehydrogenase

= 70,000)

(M.W.

at

The column

following

yeast

each

on a Sephadex

70 cm) equilibrated

dehydrogenase

(M.W.

-homoserine

sspartokinase

by gel. filtration

= 240,000),

malate

the

appropriate

calibrated

catalase

of

cm ; length

fb, 1.5

temperature

pig

weight

was determined

@ 200 column

RESEARCH COMMUNICATIONS

determination.

The molecular dehydrogenase

AND BIOPHYSICAL

liver (M.W.

= 150,000),

and cytochrome dehydrogenase

c

was

marker.

RESULTS Genetic

localization The mutation

viously

localized

duction

(8).

point

thrA in

As it

mutation.

In

as previously mutation

the

reverts

easily,

addition

the

of of

globules

the

Gif

independence

thrA -1

106M1 has been

cluster

of E.coli

it

be considered

must

complementation

showed

of

of

Analysis

homoserine

that

thrA

K12 by transas

analysis 1101

pre-

is

a

performed

a missense

cistron.

the

to

cooperativity is

n = 3 found

a first

dehydrogensse This

aspartokinase

respect

activity

was found

and HfrH.

of the

of the In

difference

106~1

with

remaining value

the

by Gif

inhibition.

activity

number

(3)

in

No detectable

tracts

carried

threonine

described

localized

Threonine

1101

of

n = 1.6, with

molecular set

their

of

the

is

between

the

measured expected

specific

in

crude

because

and homoserine

of the

dehydrogenase

catalytic

functions.

The Hill

threonine

inhibition

of

significantly wild-type

Lower

than

this the

(9).

WeiRh%.

experiments,

the

221

apparent

ex-

molecular

Vol. 60, No. 1, 1974

weights the in

of the

presence Fig.

BIOCHEMICAL

protein of

1 (part

106Ml

This

percent

in

the

amount

120

to from

presence the of

protein

I40

160 ELllTlON

80 100 VOLUME lmll

on Sephadex :

120

were

compared

profiles

of

the

I40

160

G-200.

are

activity

in shown

from

The following

crude

HfrR in presence of threonine 2 mM Gif 106Ml in presence of threonine 2 mM HfrH in absence of threonine Gif 106Ml in absence of threonine.

corresponds

However,

This

100

Seventy

RESEARCH COMMUNICATIONS

106Ml

A and B).

was eluted

dehydrogenase

and Gif

The elution

Gel filtration were analyzed A) B) C) D)

Gif

HfrH

2 mM L-threonine.

60

Fig. 1. extracts

from

AND BIOPHYSICAL

in

the

same volume

a molecular Gif

106Ml

is

of threonine crude

a lighter which

extract

of

active was eluted

weight thus

of mostly

as is

the

Gif

106Ml

homoserine in

the

222

as the

activity

350,000. in

enzyme

HfrH.

The homoserine

a tetrameric from

contains dehydrogenase

region

of

the also

form

wild-type. a small

(M.W.

corresponding

= 85,000). to

the

Vd. 60, No. 1, 1974

aspartokinase

I-homoserine

sensitive In

BIOC~EMI~L

to

threonine

the

absence

(Fig.

wild

was still

type

genase peak

activity

I monomer,

C and D).

extracted to

the

threonine,

eluted

remains

RESEARCH CO~UNICATIONS

is

no longer

inhibition.

part

corresponding

activity

dehydrogenase

of

1,

different

AND SIOP~YSICAL

elution

Whereas

as a tetramer, from

Gif

l.06~1

a molecular

sensitive

to

weight

threonine

pattern

was very

the

enzyme

from

the

homoserine

the dehydro-

was eluted

as a single

of

170,000.

roughly

The

inhibition.

DISCUSSION

The homoserine Gif from

106Ml

presents

those

Whereas

dehydrogenase

of

the

extracted

the

of

tetrameric

enzyme

latter from

presence

dissociation

is

the

mutant

threonine,

is

of

this

the

the

crude

dissociates

definitive

argument

that

usually

mutant

which

are

different

wild

type

strain

extracts,

either

in

known

a species

molecule

homoserine

of

from in

a ligand

noting

can retain

rea,ssociation

extracted

the

the the

to

strain

HfrH.

protein

absence

or in

stabilize

the

(10).

worth

The presence

from

properties

a tetramer

structure

It

extracted

seems to

indicate

dehydrogenase

can be drawn

the

MW 85,000

subunits

that

activity.

from

under

is

this the

the

monomer

However,

experiment

test

observed.

no

since

conditions

a

cannot

be excluded. The genetic lies

in

vate

the

areas inhibition

the

data

kinase

kinase

between is

indicate

region

activity, the

of the but

subunits.

consistent

that

protein.

also The low

with

the

this

it

amino

IJot only may alter

cooperativity conclusion.

223

acid

replacement it

the

does inactiassociation

of These

threonine data

point

out

Vol. 60, No. 1, 1974

the

BIOCHEMICAL

correlation

between

and dissociation the

model

An enhanced dehydrogenase enzyme results,

is

the

thrA

previously

should of

found

towards

these

which

mutants

aspartokinase is

in

mutant

threonine

(9).

to

with

globules

agreement

aspartokinase

in

interest

RESEARCH CO~UNICATIONS

with

(4).

of the

been

be of

the

proposed

dissociability

I has also

it

of

a result

desensitized

properties

alteration

ability,

structural

AND BIOPHYSICAL

strains

where

In view

of

correlate

their

I-homoserine

the

genetic

the

our

dissociation

localization

in

gene.

Acknowledgments. We are grateful to Dr. J.C. Patte for the gift of strain Gif 106Ml. We are deeply indebted to Dr. G.N. Cohen for numerous suggestions and discussions. In particular, we wish to thank Drs. B. Burr, P, Truffa-Bachi and J. Janin for their help during the preparation of the manuscript. This work has been supported by grants from the Dglbgation Gdnbrale 2 la Recherche Scientifique et Technique, The Centre National de la Recherche Scientifique (E.R. no 85) and the National Institutes of Health.

REFERENCES 1.

FALCOZ-KELLY, TRUFFA-BACHI, 28, 50-i-519.

F., P.,

JANIN, J., SAARI, J.C.; and COHEN, G.N. (1972)

2. VERON, M., FALCON-KELLY, F., Eur. J. Biochem. 28, 520-527. 3.

and SAINT-GIRONS,

THEZE, J., mi,

VERON, M., Eur. Y. Biochem.

and COHEN, G.N. I.

(1974)

J.

(1972)

Bacterial.

990-998.

4. VERON, M., SAARI, J.C., (1973) Eur. J. Biochem. and PATTE,

J.C.

VILLAR-PALASI, 38, 325-335. (1972)

J.

C.,

and COHEN, G.N.

Bacterial.

5.

:3Y;,E., .

6.

PATTE J.C., TRUFFA-BACHI, P., and COHEN, G.N. Biophys. Acts f28, 426-436 Biochlm.

224

112, (1966)

Vol. 60, No. 1,1974

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

7.

STADMAN, E.R., COHEN, G.N., LEBRAS, G., and ROBICHONSZULMAJSTER, I-I. (1961) J. Biol. Chem. 236, 2033-2038.

8.

THEZE, PATTE,

9. 10.

J., MARGARITA, J.C. (1974) J.

D., COHEN, G.N., Bacterial. 117,

BORNE, F.,

COHEN, G.N., PATTE, J.c., and TRUFFA-BACHI, Biochem. Biophys. Res. Commun. 19, 546-550.

P.

BON, C. D.E.A.,

Paris.

Facultk

aes Sciences,

225

and

133-143.

1969,

(1965)