Inhibition of rat liver phosphofructokinase-2 by phosphoenolpyruvate and ADP

Vol. 124, No. 3, 1984 November 14, 1984

BIOCHEMICAL

INHIBITION

OF RAT LIVER PHOSPHOFRUCTOKINASE-2 BY PHOSPHOENOLPYRUVATE AND ADP

Matthias Institute

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 793-796

Kretschmer

and Eberhard

of Physiological Chemistry, Leipzig, German Democratic

Hofmann

Karl-Marx-University, Republic

Received August 20, 1984 Phosphofructokinase-2 from rat liver is inhibited by phosphoenolpyruvate and ADP. Phosphoenolpyruvate reduces the maximum activity in respect to fructose-6-phosphate and ATP but does not give rise to complete inhibition of phosphofructokinase2. ADP increases the apparent Michaelis constant of the enzyme for ATP and leaves the maximum activity in respect to ATP unchanged. The apparent Michaelis constant for fructose-6phosphate is not influenced by ADP. 0 1984 Academic press, I~=. SUMMARY:

The formation 2,6-P2)

is

and degradation

catalysed

by the

bisphosphatase

(1).

bisphosphatase

activities

enzyme

are

protein

Strong

evidence which

activities

exists

are

however

is

less

inorganic

and AMP to stimulate

The enzyme

Michaelis

constant

order

of the

There

is

In

exerts

cellular

to

reconstituted

enzyme cycle

appeared

is

the

to ATP.

concentration for

allosteric the

system, studied

to inhibit

Its

this

and

formation

of F-2,6-

apparent is in

metabolite

phosphofructokinase-2 in which (5),

of

the

(4).

control.

the

ADP and other necessary

regulation

(F-6-P)

of

on one

(2) and

were reported

affinity

and

clear.

fructose-6-phosphate

integrate

phosphoenolpyruvate, enzyme

for

no indication

order

P/F-~,~-P~

high

kinase

located

The non-covalent

and citrate

p2’

the

apparently

Phosphoenolpyruvate phosphate

that

by phosphorylation

(3) processes.

two enzyme

(F-

phosphofructokinase-2/fructose-2,6-

controlled

dephosphorylation the

of fructose-2,6-bisphosphate

the actions

potential

into

regulation

a

of the

F-6-

of effecters

on this

to be investigated. 0006-291X/84 793

All

Copyright 0 1984 rights of reproduction

$1.50

by Academic Press, Inc. in any form reserved.

Vol.

124,

No.

3, 1984

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

MATERIALS

AND METHODS

COMMUNICATIONS

Phosphofructokinase-2 (IO mU/mg protein) was prepared from rat liver by isoelectric precipitation and DEAE-Sephadex chromatography as described in (6). Substrates and auxiliary enzymes were purchased from Boehringer, Mannheim (FRG). F-2,6-P2 was prepared according to (7). The kinetic experiments were carried out with 0.02 mU of phosphofructokinase-2 incubated in a reaction mixture (0.5 ml, 25 "C) containing 100 mM imidazol/HCl, 20 mM KH2POg/K2HP04, 10 mM MgC12, and 100 mM KCl. The pH 6.6, influence of the me abolites was investigated in the concentration range between 0.2 and 2.2 mM (F-6-P) and 0.6 and 5.4 mM (ATP). For the determination of F-2.6-P2 aliquots of this mixture (usually 100 pl) were removed after 10, 20 and 30 minutes and prepared for the assay of F-2,6-P2 as described in (8). The phosphofructokinase-2 activities as obtained from these data were compared with the following phenomenological equation: [F-6-P]

[ATPI

v = v-

(1) KF - 6 - p+[F-6-P1

The constants equally weighted

In the

~,,,~l+~~~~l/Ki,,,~+~ATP1

were data.

substrate

and Methods"

estimated

range

AMP and F-1,6-P2

presence

of

inorganic shows

0

the

not of

et al.

phosphate

fit

described show

(4),

but

of

using

in

"Materials

any kinetic

AMP activation

and the

dependence

effect

differs

may be due to

difference

in

ImM)

the

phosphofructokinase-2

IADPI

ImM)

Dependence of the phosphofructokinase-2 activity on the concentration of phosphoenolpyruvate (A) and ADP (B) at different F-6-P and ATP concentrations. [F-6-Pl(mM) [ATPI 5.43 2.23 0m AA

00

5.43 0.59

0.23 0.23

794

on

to the

2

1

[PEP1 1:

did

The lack

Van Schaftingen

1

square

concentration

of

Fig.

by least

AND DISCUSSION

findings

Fig.

l+[PEPl/KiiPEP

RESULTS

phosphofructokinase-2.

value.

1 +[‘EPI/KipEp

the pH

Vol. 124, No. 3, 1984

BIOCHEMICAL Table

KF-6-P

1: Constants

0.40*0.05

different

concentrations

be completely substrate

inhibited

is

ADP also

unchanged activity

2).

The analysis

of

metabolite. with

(1)

The inhibition phosphoenolpyruvate

increases

to F-6-P

activity

the

constants

K. 5 value. .

IB)

shows that

(Fig.

in Table

be inhibited to the 1.

by be of physiological

ImM)

IATPI

Effect of ADP on the at varying F-6-P (A) A [ATPI tADPI 0 0 5.43 0 1 .lO

the

2

1

0 0

presented

might

the maximum

quantitatively

of phosphofructokinase-2 and by ADP

constant

to ATP remains

ADP decreases

fit

the

to

Michaelis

may completely data

for

In contrast

curves

low

1).

but does not affect

The kinetic

[F-WI

2:

constant

in respect

the ADP velocity

cannot

even at

apparent

hand,

and ADP at

The activity

mM (Table

the

On the other

0

Fig.

0.3OkO.02

phosphofructokinase-2.

the phosphofructokinase-2

equation

of 0.30

the maximum activity

in respect

by this

2.3OiO.30

The characteristic

it

(Fig.

K iiPEP

of phosphoenolpyruvate

order

inhibits

ATP while

K iPEP

by phosphoenolpyruvate,

in the

phosphoenolpyruvate for

(l)(mM)

of ATP and F-6-P.

concentrations.

inhibition

equation

0.16iO.03

on the concentration

activity

of

K iADP

KATP

0.09*0.01

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ImM)

phosphofructokinase-2 activity and ATP (B) concentrations B [F-6-P](mM) [ADPI A A

0.59 0.59

195

0 1 .lO

2.23 2.23

Vol. 124, No. 3, 1984

significance. (9)

are

The levels

in

the

constants. level

in

with

the

the

present

might

metabolic

range

rat

of

of

that

by inhibition

states

at

preferably

by the

ADP level. low oxygen

adenine

is accompanied The results

F-2,6-P2

We hypothesize supply

cycle nucleotide

system.

2.

3. 4.

5. 6. 7. 8.

9.

Van Schaftingen, E., Davies, D.R., and Hers, H.-G. (1982) Eur. J. Biochem. 124, 143-149. Pilkis, S.J., Walderhaug, M., Murray, K., Beth, A., Venkataramu, S.D., Pilkis, J., and El-Maghrabi, M.R. (1983) J. Biol. Chem. 258, 6135-6141. Mieskes, G., Brand, I.A., Soeling, H.-D. (1984) Eur. J. Biochem. 140, 375-383. Van Schaftingen, E., Hers, H.-G. (1981) Biochem. Biophys. Res. Commun. 101, 1078-1084. Eschrich, K., Schellenberger, W., and Hofmann, E. (1983) Arch. Biochem. Biophys. 222, 657-660. Van Schaftingen, E., Davies, R., and Hers, H.-G. (1981) Biochem. Biophys. Res. Commun. 103, 362-367. Van Schaftingen, E., and Hers, H.-G. (1981) Eur. J. Biochem. 117, 319-323. Van Schaftingen, E., Lederer, B., Bartrons, R., and Hers, H.-G. (1982) Eur. J. Biochem. 129, 191-195. Hue, L. (1982) Biochem. J. 206, 359-365.

796

of

level in that

the

REFERENCES I.

F-2,6-P2

phosphofructokinase-2

phosphofructokinase-I/fructose-I,6-bisphosphatase regulated

of the

which

decreasing

of the

increasing

arising

decrease

anaerobiosis

the

hepatocytes

inhibition

of ADP and AMP (9).

suggest

the

in

characteristic

under

increase

study

intermediates

an unexpected

hepatocytes

wellknown

these

of their

Hue reported

be caused

consequence

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

is

in