Fructose-1,6-diphosphatase of acinetobacter : Inhibition by ATP and citrate

Vol. 37, No. 2, 1969

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FRUCTOSE-1,6-DIPHOSPHATASE OF ACINETOBACTER : INHIBITION

BY

ATP AND CITRATE Antony Department

J. Mukkada and Emily

J. Bell

of Biological Sciences and the Graduate of M icrobiology, University of Cincinnati Cincinnati, Ohio 45221

Division

Received August 20, 1969 Summary Fructose-1,6-diphosphatase (FDPase) from a species of Acinetobacter was partially purified and some of its properties studied. The enzyme resembles other FDPases investigated in its dependence on a divalent metal like Mg++ or Mn++, high affinity for the substrate, stimulation by EDTA and inhibition from by high substrate concentrations. However, it differs most other FDPases in its insensitivity to AMP and inhibition by ATP and citrate. Introduction Fructose-1,6-diphosphatase phosphohydrolase, strongly 1965).

EC 3.1.3.11)

inhibited However,

Taketa

and Pogell

inactivation

enzyme in Candida utilis they

contaminant.

attributed In the slime

fructose-1,6-diphosphatase AMP or dAMP (Rosen, the presence negative

9957, reclassified

that

ATP

of the enzyme from rabbit

by high

some inhibition

of the

concentrations

to the traces

of ATP and

of AMP present

mold Polvsphondvlium is not appreciably In this

Acinetobacter

sp.

as Acinetobacter

1968) which

is

by ATP (Atkinson,

is strongly 340

as a

pallidum, inhibited

communication

of a fructose-1,6-diphosphatase

bacterium

and Stanier,

1966).

l-

of sources

(1963) reported

(1965) noted

Rosen, -et al.

ADP which

from a variety

by AMP and activated

causes a reversible liver.

(D-fructose-1,6-diphosphate

by

we report

in the gram

(Mima polymorpha

ATCC

by Baumann, Doudoroff inhibited

--in vitro

by ATP

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 37, No. 2, 1969

and to a lesser

extent

by citrate

while

being

relatively

unaffected

by AMP. Materials The organism

and Methods

was grown on a simple

described

earlier

(Bell

harvested

at the m iddle

and Yarus,

1966).

The enzyme was partially

by lactic

fractionation,

precipitation through

purified

according

(1966) by measuring

nicotinamide

adenine

temperature

at 340 mp.

ammonium sulfate

(2.0 mg/ml)

the rate phosphate

The standard

M;

reaction

0.2 M , pH 9.5;

was adjusted

diluted

12 m inutes.

to 3.0 m l with

amount which

water.

The specific

converts

to fructose-6-phosphate was determined

Fructose-1,6-diphosphate

contained:

MgC12, 0.1 M ;

volume

was initiated after

were measured between 6 and of the enzyme is expressed

protein.

A unit

1 m icromole

of enzyme is the

of fructose-1,6-diphosphate

in 1 m inute

at room temperature.

by the method of Lowry,

glucose-6-phosphate

(5.0 mg/ml);

The final

The reaction

activity

(98%

isomerase

0.01 M ; and 0.1 m l

(fructose-1,6-diphosphate)

and maximum rates

per m illigram

m ixture

dehydrogenase

enzyme preparation.

the substrate

equilibration

sample absorbance

0.03 m l

0.06 m l sodium fructose-1,6-diphosphate,

of

(NADP) at room

0.003 m l each of phosphohexose

and glucose-6-phosphate

of a suitably

fraction

of reduction

model 2000 multiple

buffer,

0.03 m l NADP, 0.05

isomerase,

(40 fold)

to the method of Rosen, Rosen

dinucleotide

in a Gilford

0.60 m l glycine

Protein

in the

The enzyme was assayed

and Horecker

as units

were

and passage of the 80% ammonium sulfate

spectrophotometrically

by adding

medium

The cells

subsequent

a Sephadex G-25 column.

recorder

salts

of the log phase and ruptured

French Press. acid

m ineral

pure),

(1951).

NADP, phosphohexose

dehydrogenase, 341

et al. --

AMP, ADP, ATP,

Vol. 37, No. 2, 1969

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

EDTA and citrate St.

Louis.

were purchased

Before

addition

ATP, EDTA and citrate

to the assay system,

Growth on various acetate,

not affect stitutive

succinate

Like

also

stimulated

of the enzyme,

different

requires

inhibited response

at high

The curves

cant

decrease

takes

results.

about

inhibitor

ATP and citrate

observed

it

five

about

fold,

results

ATP, ADP,

2.0 pmoles

40-50% reduction to give

ATP although

organisms

it

is

(in

comparable less

(Atkinson,

effective. is a potent

1965).

to bind Mg++ which

the concentration

to determine

Since is essential

whether

the

of Mg++ or are It

was observed

of Mg++ in the reaction

of the enzyme could 342

a 3.0

in activity,

to AMP which

on the enzyme itself.

the activity

the addition in a signifi-

are due to the complexing

effect

by increasing

m ixture

While

was necessary

inhibitions

due to a direct that

1 and 2 show that

have the capacity

the reaction,

like

the

different.

insensitive

in most other

is at

However,

modifiers

5-7 pmoles of citrate

Our enzyme is relatively

(Mg++ or Mn?,

concentrations.

causes nearly

ADP resembles

0.009-0.013.

at 0.1 - 0.2 m M of FDP), but

to the assay m ixtures

m l assay m ixture)

of the

the enzyme from our

cation

of activities

in activity.

is con-

of EDTA and is active

marked A in Figs.

of ATP or citrate

it

activity

sources,

a divalent

is quite

ethyl - does

that

is about

of the enzyme to allosteric

AMP, and citrate

for

substrates

substrate

lactate,

suggesting

The specific

by low concentrations

low (maximum rates

- pyruvate,

and fructose-1,6-diphosphate

the FDPases from most other

organism

it

sources

the organism.

enzyme in these

AMP, ADP,

and Discussion

carbon

the level for

Co.,

were neutralized. Results

alcohol,

from Sigma Chemical

be restored

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 37, No. 2, 1969

gmoles

ATP 5 1

1

I

I

4

2

OI 01 0,

I

6 Time

7 I

6 1

8 (mln)

IO

20

30

of FDPase by ATP. The enzyme was assayed Figure 1. Inhibition Curve A shows spectraphotometrically as described in the text. the effect of increasing concentrations of ATP while curve B shows the effect of preincubating the enzyme with ATP (2.0 pmoles/3.0 m l assay m ixture) for different periods of time. Concentrations of ATP shown represent the amounts in 3.0 m l assay m ixtures. 8 I

umoles I2 I

Citrate 16 I

20 I

24 I

26 I

I

80

20

I

1

I

2

4

I

I

6 Time

0 tminl

The enzyme was of FDPase by citrate. Figure 2. Inhibition Curve assayed spectrophotometrically as described in the text. A shows the effect of increasing concentrations of citrate while curve B shows the &fect of preincubating the enzyme with citrate (5.0 pmoles/3.0 m l assay m ixture) for different lengths Concentrations of citrate shown represent. the amounts of time. in 3.0 m l assay m ixtures. 343

Vol. 37, No. 2, 1969

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

to 90% of the control

which

suggested

that

Mg++ may be the cause of the inhibitory Subsequent

experiments

preincubation citrate

(Table

cantly

greater

measurements

degrees

the same.

Table

or citrate

addition

30 m inutes,

30 m inutes

results

than those

immediately

after

1 also

shows that

in signifiobtained

adding

the effect

to the enzyme preincubated

is essentially

same as without

the

ATP or

of ATP and Citrate

Assayed immediately

Mg++ for

prior

Relative after

addition

with

MgCl2 for

Preincubated

with

ATP for after

Preincubated

with

MgC12

Preincubated

with

citrate

ATP

incubation

on FDPase actiyity

Preincubated

Assayed immediately

case are

of either with

when

1

Treatment

Assays upper while on the

m l) or

the amounts of magnesium in either

Table Effect

showed that

ATP (2.0 pmoles/3.0

of inhibition

are initiated

although

citrate,

for

ml)

of

effects.

11, however,

of the enzyme with

(5.0 pales/3.0

the complexing

30 m ins.

activity (%I 47.1 43.0

30 m ins.

11.9

addition

50.0

for

30 m ins. for

30 m ins.

48.4 14.1

The were run at alkaiine pH as described in the text. half of the Table shows the effect of 2.0 pmoles of ATP the lower half shows the effec,t of 5.6 pmoles' of citrate relative activity of the enzyme. 344

Vol. 37, No. 2, 1969

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of the enzyme with assays

the cation.

It

the amounts of magnesium,

in each case and the time citrate

is the same.

may be noted that

ATP and citrate

of exposure

Mg++ to the reaction

distinct

before

inhibitory

by the results

incubated

with

effect

overnight

to remove free

the usual

manner using

enzyme as a control.

for

inhibitory

of

in which

these

was dialyzed

and then assayed

was found that

in

sample of

the ATP and citrate

activity

the control

of

the enzyme

but dialyzed

enzymes showed a residual

9.0% respectively

that

the

on the enzyme which

ATP and citrate

It

the addition

30 m inutes

an untreated

if

This was further

of experiments

ATP or citrate

noted

indicates

from Mg++ complexing.

confirmed

treated

of magnesium to ATP or

clearly

m ixture,

compounds have a direct is quite

are constant

The very marked inhibition

enzyme is exposed to ATP or citrate

in all

clearly

of about 6.0 and demonstrating

the

of these compounds on the enzyme protein

effects

itself. The inhibition time

by ATP and citrate

of incubation

Figs.

with

1 and 2 show the

the enzyme. effect

the absence of Mg++) with varying

periods

The curves

of incubating

ATP and citrate

prior

ted.

substrate

would give

for

in assays

of titrate/3.0

phenomenon is clearly

of any structural

relationships

(fructose-1,6-diphosphate) suggests

which,

40-50% inhibition

m l and 5.0 pmoles

dependence of this

(ATP and citrate)

respectively

In these experi-

incubation,

The lack

the enzyme (in

of assay in each case.

(2.0 pmoles of ATP/3.0 The time

marked B in

Magnesium was

ments we used an amount of the inhibitors without

upon the

up to 30 m inutes.

of time

added at the time

is dependent

that 345

m l).

demonstrabetween the

and the inhibitors these

interact

with

the enzyme

Vol. 37, No. 2, 1969

at

an

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

allosteric

site

to determine

sites.

or

the nature

As in Escherichia

Studies

of these

coli

(Fraenkel,

et al., --

in our organism

the insensitivity

to AMP and susceptibility

make the FDPase of

The physiological to note that

significance

inhibition

the catabolic

is also

of Acinetobacter

1966) the fructose-

constitutive.

Acinetobacter

quite

is not clear.

It

by ATP and citrate

anabolic

FDPase.

It

has both a catabolic

However,

to ATP and citrate distinctive. is

interesting

is characteristic

enzyme phosphofructokinase

not of the usually

directed

sites.

1,6-diphosphatase

inhibition

are being

(Atkinson,

1965) and

may be that

the FDPase

and anabolic

role.

Acknowledgment This work was supported Research Grant Allergy

AI-08050

by U.S.

Public

Health

from the National

and Infectious

Institute

Service of

Diseases. References

Atkinson,

D.E.,

150, 851 (1965).

Science,

Baumann, P., Doudoroff, 2, 1520 (1968). Bell,

E.J.

and Marus,

and Stanier,

R.Y.,

J. Bacterial.,

91,

M.

A.,

Fraenkel, D.G., Pontremoli, Biochem. Biophys., 114,

S. and Horecker, 4 (1966).

Lowry, O.H., Rosebrough, N.J., Farr, J. Biol. Chem., 193, 265 (1951).

A.L.

B.L.,

Rosen, O.M.,

114,

Biochem.

Biophys.,

J. Bacterial., 2223 (1966). B.L.,

Arch.

and Randall,

Rosen, O.M., Rosen, S.M. and Horecker, Biophys. 112, 411 (1965). Arch.

Arch.

R.J.,

Biochem.

31 (1966).

Rosen, O.M., Rosen, S.M. and Horecker, B.L., in Methods -in Enzymology IJ, W.A. Wood, ed., Academic Press, New York, 1966, p. 632. Taketa, K. and Pogell, 12, 229 (1963).

B.,

Biochem. 346

of

Biophys.

Res. Commun.,