Identity in escherichia coli of carbamyl phosphokinase and an activity which catalyzes amino group transfer from glutamine to ornithine in citrulline synthesis

BIOCHEMICAL

Vol. 18, No. 4, 1965

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

IDENTITY IN ESCHERICHIACOLI OF CARBAMYLPHOSPHOKINASE AND AN ACTIVITY WHICHCATALYZESAMINO GROUPTRANSFERFROMGLUTAMINE TO ORNITHINE IN CITRULLINE SYNTHESIS* Sumner M. Kalman,,Patricia H. Duffield and Thomas Brzozowski Department of Pharmacology, Stanford University School of Medicine, Palo Alto, California

ReceivedDecember31,

1964

Pierard and Wiame (1964) showed recently of wild

type Escherichia

coli

were able to catalyze

of an amino group from glutamine citrulline activity Previously

formation.

that extracts

to ornithine

Presumably this

transfer

in ATP-mediated

is the same type of

discovered by Levenberg (1962) in Agaricus bisporus. it had been considered that carbamyl phospho-

kinase was the sole enzyme activity

responsible

for carbamyl

phosphate synthesis, NH2 / \ OP03H-

E

CO2 f NH3 + ATP-O=C m*

in the step leading also citrulline

to carbamyl aspartic

acid formation

and

biosynthesis

deals with purification

in -E, coli. The present report of the glutamine-dependent activity

(GDA) from --E, coli B, and its phosphokinase (CPK).

apparent identity

to carbamyl

*Aided by grant #E-345 from the American Cancer Society 530

Vol. 18, No. 4, 1965

EIOCHEMICAL

Evidence for identity

AND BIOPHYSICAL

includes

RESEARCH COMMUNICATIONS

the following

observations:

1. Purification.

Wild type --E. coli B was grown in minimal medium plus glucose (Davis and Mingioli, 1950) with addition

of 3% casamino acids,

the exponential as indicated cedure will purification

Cells were harvested during

phase of growth, sonicated,

in Table 1. be included

Details

glutamine-dependent

of the purification

in a later

of approximately

publication.

75-fold

activity

and then purified Overall

was achieved for the

and about 36-fold

bamyl phosphokinase activity.

pro-

for the car-

Ratios of GDA to CPK activity Table 1

Purification

Procedure for Carbamyl Phosphokinase from AE. coli

The assay mixture for glutamine dependent citrulline synthesis contained per ml 100 umol Tris pH 8.5, 24 urn01 MgCl2, 12 ~01 L-ornithine HCl, 24 ~01 ATP, 6 u,mol Lglutamine, 30 ~01 KHC03, and excess ornithine transcarbamylase from S. faecalis. The assay mixture for ammoniadependent citri.irlXne synthesis contained per ml 200 ~01 Tris pH 8.5, 20 ~01 MgC12, 10 ~01 L-ornithine HCl, 20 ~01 ATP, 100 u,mol (NH4)2CO3, and excess ornithine transcarbamylase from S. faecalis. Incubations were carried out at 370 for 15 mic the reaction was stopped with an equal volume of 10% TCA. Citrulline was determined by a modification of the Archibald method (1944). One unit of either enzyme represents the ability to form 1 ~01 of citrulline per hr. A

B

S.A.

S.A.

0.40

0.42

Protamine sulfate ppt'n (5 mghl) Annnoniumsulfate ppt'n (35-60%)

1.13

.50

2.26

4.60

2,18

2.11

Sephadex G-200

7.60

4.20

1.90

30.00

14.40

2,08

-~ Step Cell extract

Protamine sulfate (.Ol-.05 mg/ml)

ppt'n

A = GDA; B = CPK 531

A/B

0*95

Vol. 18, No. 4, 1965

changed (Table

BIOCHEMICAL

once during 1).

This

purification;

change in

to any appreciable

extent

remains

kinase

in

and carbamyl

Harmon,

1962)

greater

increase

CPK at

this

discrepancy ties

the

cell

extract

that

by protamine

supernatant

at

stage

specific

this

activity

of the

the

purification

degree

shown in Table

activity

other

showed a remarkably

constant

ratio,

many procedures

reported

in

not

to alumina

C-y and calcium

in various

media

(see

step

adsorbed

The activity

step

has both

aceto-

(see Grisolia

and

activity.

The

of GDA relative accounts

of purification In all

1,

is not

sulfate.

no glutamine-dependent in

sulfate

to be due to the presence

phosphokinase

but

in

the

RESEARCH COMMUNICATIONS

see protamine

appears

of a phosphokinase

which

AND BIOPHYSICAL

for

for steps This

the

table,

phosphate

gel,

to

the

the

the

two activi-

two activities

was observed such

2-fold

in

as adsorption

and chromatography

below).

AAA 0.D2.53 . . . GDh 300 CPK

Figure

1.

Chromatography

on Sephadex

G-200.

Five mg of purified material was layered on a 50 x 1 cm column made up with 0,02 M phosphate buffer, pH 6.0, containing 20% glycerol. Elution was carried out with the same solution. Ratio of GDA:CPK for the starting material was 2.3. 532

Vol. 18, No. 4, 1965

BIOCHEMICAL

AND BIOPHYSICAL

2. Chromatopraphy. activities

application

the

presence

Sephadex

to columns

from

3, Co-sedimentation. to a sucrose

traveled

together

as a single

acetate

apparatus.

tudinal

section

kinase

and the

as a single

lower

than

to the

carbamyl

compared

the

after

sufficient a longi-

carbamyl

activity

observation

that

was applied

by staining

Both

phospho-

moved

was repeated

In the

pH 8.7

phosphokinase

observation

alkaline

at pH 4.5,

strips

activity;

GDA is relatively

together

GDA was

this

corresponds

unstable

in

solutions. Electrophoresis

and 8.6.

In each

activities,

in case

starch

gel was carried

out

at pH 7

a single

band contained

both

enzyme

In addition,

performed

with

gel

no separation Results

of

heated

One band

When a sample

to starch

electrophoresis

a sample

GDA was destroyed.

Table

electrophoresis

a Shandon were

at pH 8.7.

was

two activities

in

glutamine-dependent

pH 6, and also

the

material

material

strip.

This

the

column.

as determined

of each

band.

1 shows

The purified strips

had occurred

(2M),

peak.

The two activites

migration

hydrochloride Figure

gradient

4. Electrophoresis. to cellulose

These

When the purified

density

two

DEAE-cellulose

of guanidine

a G-200

the

failed.

containing

and hydroxylapatite.

of elution

applied

to resolve

techniques

or absence

G-200,

pattern

ties.

attempts

by chromatographic

included in

All

RESEARCH COMMUNICATIONS

of

the

electrophoresis of

starch

the gel

enzyme

at

60'

contained purified in

2. 533

C until both

starch most

gel was of

residual

the activi-

enzyme was subjected the presence

activities

electrophoresis

in

of 2M formamide

was demonstrable. are

presented

in

Vol. 18, No. 4, 1965

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Table 2 Starch Gel Electrophoresis The gel column was that described by Smithies (1955), 20 mm wide x 250 mm long x 6 mm deep. The experiments were carried out for 3 to 4 hrs with a gradient of 400 volts. Protein bands were located on a thin slice of starch gel stained with amido black. Corresponding bands of the unstained starch were divided and incubated in the two assay mixtures described in Table 1. Note that this particular preparation had a higher GDA/CPKratio than the one described in Table 1. At pH 7 the buffer system employed was .076 M Tris-citrate (starch) and .3 M borate (bridge solution) (Poulik, 1957). At pH 8.6 the buffer system was 0.01 M barbital (starch) and 0.1 M barbital for the bridge solution. Ratio,

GDA:CPK

Starting

PH 7

Radio, GDA:CPK

material

Starch band

3

2.2

7 + 2M formamide

1.2

1.1

8.6

3

1.8

5. Reaction to inhibitors. sensitive

to the same set of inhibitors,

some differences especially

Both activities

group from glutamine glutarate. are sensitive

The GDAwas

to azaserine and potassium fluoride,

both of which have been used to inhibit Both reactions

although there were

in degree of depression.

sensitive

were

transfer

in other enzyme studies

were sensitive

These results

of an amino

(Meister,

1962).

to the presence of a-ketoare shown in Table 3, Both enzymes

to parachloromercuribenzoate. 534

Vol. 18, No. 4, 1965

BIOCHEMICAL

AND 8lOPHYSlCAL

RESEARCH COMMUNICATIONS

Table 3 Inhibition

of Enzyme Activities

Preincubation with each of the inhibitors, a-ketoglutarate, azaserine, and potassium fluoride was carried out at 370 for 10 min. Activities were assayed as described in Table 1. Inhibitor

Cont.

Control a-ketoglutarate

6 x lO-3 M

% inhib GDA

% inhib CPK

*GDA

*CPK

100

40

14

0

84

100

azaserine

10-4 M

64

40

36

0

KF

10-3 M

60

40

40

0

*Activities

are expressed as units

6. Treatment with thiol at a concentration

reagents.

Dimercaptoethanol

of .Ol M did not produce resolution

any of the preparative tol,

per ml

10-3 4 (Cleland,

steps.

Incubation

in

with dithioerythri-

1964), and subsequent chromatography

on Sephadex G-200 did not separate the two activities. There were data that did not show completely behavior for the two activities. activity

was relatively

fluoride.

dependent activity

The carbamyl phosphokinase

insensitive

In addition,

parallel

to azaserine and potassium

it was found that the glutamine-

was unstable at room temperature,

at 0' C,

and at -20' C in the presence of .OOl M ethylenediaminetetraacetic

acid and .Ol M mercaptoethanol.

glycerol

to all

solutions

under these conditions Purification ditions.

of 20%

markedly improved stability.

Even

GDAwas unstable at alkaline

was carried

kinase activity

The addition

out at pH 6.

was relatively

Extreme lability

The carbamyl phospho-

stable under ordinary

of the glutamine-dependent 535

pH's. conactivity

Vol. 18, No. 4, 1965

in

the

BIOCHEMICAL

absence

of glycerol

in purification before

lag

studies

it did

yield

(carbamyl

phosphokinase)

a control

sample

for

a similar

This

report

presents

phosphokinase

with

of an amino

group

citrulline,

an enzyme

Pierard

changed

from

about

in

from

specific

chromatography behavior

in

in

an enzyme

that

glutamine

formamide,

similarity

in response

Several

have

An example

isolated

appeared

media,

to lose

it

retained

which the

in

guanosine

own earlier CPK activity The data

had been did

identity

of car-

catalyzes

transfer

synthesis

of

reported

in E . coli

includes

by

a parallel

purification,

coidentical

no resolution

when treated

with

reagents,

and some degree

of

catalyze

capacity

the

the its

ability

M for

to inhibitors.

is

During

that

co-sedimentation,

amino

from

process

to utilize

This

attempts persisted presented

experience

at purification in

536

aminase

Aerobacter this

enzyme although

of the is

that

amino

similar

of GDA whereby

the absence

suggest

(Meister,

glutamine

to use NH3 as a source

5'-phosphate,

transfer

5'-phosphate

(1957)

purification

ability

group

to use NH3 also

xanthosine

by Moyed and Magasanik

aerogenes.

the

during

or thiol

enzymes

glutamine

in first

electrophoresis,

NH3

1 x 10-3

the

The evidence

several

km for

(GDA activity)

for

activity

The

of inactivation.

activities

guanidine,

1962).

glutamine

time

denaturation

the

a sample

evidence

and Wiame (1964).

increase

from

that

degree

the

of inactivation.

fact

M for

difficulties

and for

Heat

by the

The Km for

change

(1962)

curves

to 6 x 10-2

75% inactivated.

for

in bacteria.

parallel

was complicated

bamyl

by Levenberg

was discovered not

RESEARCH COMMUNICATIONS

may be responsible

mentioned

procedure

not

AND BIOPHYSICAL

to our only

of glycerol. two enzyme

group

activities

the

BIOCHEMICAL

Vol. 18, No. 4, 1965

which

catalyze

properties the

the

fraction

which

contained

single

about

30-50%

glutamine,

for

two sites

differ

it

bound

It

our

together

present

in

their

It

is

active

that

sensitivity

or share

very

and the

to chemical

that

indicate

a NH3 and

sites,

further

In any case,

data

the

patterns)

is probable

possible

in

to represent

of two substrates,

be successful.

proteins

protein

are

electrophoretic

has different

somewhat

will

two distinct tightly

which

treatments,

separation

from

either

phosphate

appeared

activities.

enzyme may utilize

and physical

Of the

(as estimated both

RESEARCH COMMUNICATIONS

of carbamyl

protein.

material

active

at

formation

of a single

purified

AND BIOPHYSICAL

that

if

they

similar

attempts there

must

are be

physical

properties.

REFERENCES Archibald,

R. M.,

Cleland, Davis,

W, W.,

J.

Biol.

Biochem.

2,

B. D. and Mingioli,

Grisolia, 482 Levenberg,

S. and Harmon, (1962). B.,

J.

Biol.

Chem. 156, 480

(1964),

E. S.,

J.

121 (1944).

Bact.

PO, Biochim. Chem. 237,

60,

Biophys. 2590

17 (1950). Acta

(1962).

Meister, A. in Boyer, P. D. et al., ed,, The Enz Vol. IV, Academic Press, New York (1 e Moyed,

H. S. and Magasanik, (1957).

Pi&ard, l5, Poulik, Smithies,

A. and Wiame, 76 (1964). M, D., O.,

J.

Nature

180,

Biochem.

J,

B., M.,

J.

Biochem.

1477 6l,

Biol.

(1957).

629 (1955).

537

59,

es

Chem. 226, Biophys.

Res,

351 Comm.