End-product inhibition of yeast phosphofructokinase by ATP

BIOCHEMICAL

Vol. 12, No. 2, 1963

EED-PRODUCT IEHIBI!J!ION

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

OF YEAST PHOSPHOFRUCICOKIRASE BY AU? '

E. Vi5uela,2

Maria IL S~&W,~

and A. Sol8

Department of Enzymology, Instituto MarafXdn, Centro de Investigaoionea Bioldgicas, C.S.I.C., Madrid, Spain

Received

May

20,

1963

Some relation reaction twenty

to the Pasteur years,

colysis

effect

even after

became centered

acceptor. ed for

of the phosphofructokinase

Indirect

and for

yeast

has been suspected

on the phosphate for

(Iynea

et al.,

Maitra

and Steinberg,

1963),

1959),

and muscle (Park et al.,

1961).

Direct

and its

counteraction

ly reported

for

and Mansour,

for

lity

Fasciola

it

ascites

Research

(Negelein,

by A!l!P competitively

not supported

by

Newsholme and Randle, has been recent1962).

Nothing

was

where the Pasteur

1936),

In addition

there

was a possibi-

than the muscle enzyme.

below show that with

of the Comissria

1957)

and muscle PFK (Mansour

could be less unstable

Fellow

has been claim-

of PFK by excess ATP

hepatioa

found.

of glg-

tumor (Lonberg-Holm,

1961;

This work was supported by a research Public Health Service (RC-8041). 2

over

phosphate

and Potter,

1959; although

inhibition

The data presented inhibited

relation

Passonneau and Lowry,

1962;

was originally that

and/or

by severalmetabolites

known on the PFK of yeast effect

this

systems (Aisenberg

evidence

for

the emphasis on the control

evidence

reconstructed

(PFK)

yeast PFK is

fructose-6-phosphate grant

from U.S.

de Proteocicn

Escolar.

BIOCHEMICAL

Vol. 12, No. 2, 1963

(FSP),

in such a way that

logical with

AND BIOPHYSICAL

concentrations the increase

the square

the normal rauge

the activity

of F6P concentration.

end-product

It

and increases is suggested

as the end product

irreversible

with

that ATP

of the pathway whose

step is the PFK reaction,

inhibition

of physio-

of the enzyme decreases

of ATP concentration

may be considered first

within

RESEARCH COMMUNICATIONS

and that

by ATP can act as a feed-back

oontrol

in glycolyaia.

The effect PFK activity Fig.

3 at a given

1. If

Within

plot

kinetics.

respect

3

is no inhibition

the same range. of F6P corresponds

lines

with respeot lines

with

(B). The reaction

if

ATP is substituted the double

respect

by GTP (Fig. plots

reciprocal

to the concentration to the square

becomes first

reciprocal

the

to second

2, the double

to F6P at very high F6P/ATP ratios

conditions, (Fig.

by GTP, there

As shown in Pig.

and straight

of the latter

of F6P is shown in

a wide range of UP concentrations,

gave parabolic

of F6P (A),

ranges

within

of the concentration

order

of &l!P on yeast

concentration

ATP is substituted

by excess nucleotide effect

of the concentration

order

with

and over wider 3A). Under these

give

parallel

lines

3).

Yeast PFK was obtained by extracting dried brewers yeast (Anheuser and Busch, "for enzyme work") with 6 volumes of 0.1 M KHCO3 - 1 mltl CH -CH2SH - 1 mM EDTA. The fraction precipitated between i 0 and 60 per cent saturation of (NE4)2SO4 was dialyzed against several changes of 0.05 Y potassium phosphate - 5 mM MgC12 - 1 m&I CH3-CH2SH - 1 ti EDTA, pH 7.5. This PFK preparation is free of ATPase and is stable within the assay conditions in this work. 141

Vol. 12, No. 2, 1963

BIOCHEMICAL

AND BlOPHYSlCAi.

RESEARCH COMMUNICATIONS

t3TP Oo3 d 2 t z 3

/-

0.2 /p \\

O-l !

NP 1 Q6

I 1

I 1.5

.I 2

(XTP) llw

Fig.

1. Yeast phoaphofructokinaae activity GTP, at 0.9 mM fructose&-P.

with ATP and

PFK activity wae assayed by following the decrease in optrcal density at 340 mfl in the presence of excess aldolase, trioae phosphate ieomerase, and LO(-glycerophosphate dehydrogenaae (Boehringer), 0.1 mM DPKH, 0.05 M Tris pH 7.5, 5 m&l CH3-CH#H, 10 mM MgC12, P6P (obtained by treating glucose&-P with glucose phoaphate isomeraae and assuming the concentration of F6P in the equilibrated mixture to be l/4 of the total ester), and ATP or GTP a8 indicated in the figures. The reaction was started by the addition of the PFK preparation. Kate is expressed a8 )unolea of Hub&rate tranaformed per min per mg protein.

I

2

3

WFbPh

4

5

sol5m25

lo-’

fl/F6Pho-.

Fig, 2. Effect of the concentration of fructose-6-P phoephofructokinaee activity at the concentrations ATP-Mg indicated in the figure, in millimolea/l. Other assay conditione as in Fig. 1.

No appreciable (at inhibitory -3',5'-AMP,

levels

effect

on the activity

of ATP) was observed

AMP, ADP, or FDP (using

of yeast PFK

with Pi,

in the latter

on of

cyoliccase the

Vol. t2, No. 2, 1963

pyruvate range

BIOCHEMICAL

kinase-lactic

dehydrogenase

of concentrations

I

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

method)

within

the

used by Passonneau and Dowry (1962).

I

I

1

2

I 3

I 4

(l/F6P)xlo-3

WGTP)xlo-’

Pig. 3. KXnetice of the phosphofructokinase reaction in non-inhibitory conditions. Assay conditions as in Pig. 1, at the concentrations of GTP and F6P indicated in the figure.

DISCTJSSIOB The strong

inhibition

of yeast PFK by excess ATP,

but not by excess GTP (see Fig. has a regulatory

binding

site

for

substrate

site.

occupancy

of the regulatory

occupancy

of the sugar substrate

order

kinetics

Competition

l),

suggests ATP different

with F6P further

with respect

site

panty

that

for

ATP, or simply

of a bridging

site.

suggests

Moreover,

site

involve

ATP regulatory

those of F6P in two active

with

respect 1963).

from the that

with the second

to F6P which accompany inhibition

with Sols,

the enzyme

by ATP interferes

could depend on a second regulatory with

that

for

F6P interfering

a dimer in which oocusite

sites.

could

be competitive

Second order

kinetics

to F6P is shown also by muscle PPK (Vifiuela The second order

kinetics 143

in relation

and

to some

BIOCHEMICAL

Vol. 12, No. 2, 1963

regulatory

inhibitions

(Chsngeux,

may have a general

1962)

AND BIOPHYSICAL

in terms of polymerized

lation,

possibly

that

simultaneously

Similar

kinetics

on a given

llhia Its

pathway after

irreversible

site

with

over a wide range and $018,

(Moyed and Umbarger, in this tive

with

case the end product

enzyme. !l!he fact

ing A!CP within trations

that

leads to A!l!P production. Inhibition

in normal

biosynthetic

pathways

the additional

novelty

PFK activity

decreases

range

yeast suggests

that

this control

In aerobiosis,

Such a feed-back

the rate

of the A!l!P-producing

all

of net disposal

rate

mediates

(for

glycolytic

of A!I!P, without

an illustration

see Holzer, 144

of the sensiwith

increas-

end-product mechanism

the increased

of A!L'P per mole of hexosemonophosphate

brake the PFK reaction.

that

of ATP and F6P concen-

by A!CP can act as a feed-back yeast glycolysis.

inhibition

is also a substrate

the physiological

in fermenting

inhibition yield

1962),

of the cross-road.

step is the PFK reaction.

for

of

1963).

as the end product

specifically

established

1953).

a muscle PFK prepa-

of PFK by A!l?P would then be a case of end-product in the sense well

condi-

(Alberty,

the hexosemonophoaphate

is the pathway that first

active

(VifXuela

ATP may be considered glycolytic

levels, F6P and A!l?P, are not

to A!l?P inhibition,

ATP and F6P concentrations

regu-

of the

of yeast PFK in non-inhibition

have been observed

desensitized

as mechanism,

sensititity

the two substrates,

present

and Pardee,

enzymes. For metabolic

enzyme to changes in metabolite

suggests

ration

far

As

in terms of increased

The kinetics tions

Gerhart

1961;

significance.

perhaps regulable

RESEARCH COMMUNICATIONS

would further

control

could adjust

pathway to the overpiling 1961).

up of inter-

BIOCHEMICAL

Vol. 12, No. 2, 1963

AND BIOPHYSICAL

Passonneau and Lowry (1962) large

increase

in PFK activity

when the ATP concentration ficance

is the fact

competitive

with

that

have emphasized

the

in muscle that may be expected

falls,

Of possible

the inhibition

greater

signi-

of PFKa by ATP is

the square of the F6P concentration.

property

opens a way for

activity

by the large

in stimulated

RESEARCH COMMUNICATIONS

automatic,

increase

muscle,

before

drastic

forcing

in F6P formation any marked fall

This of PFK

that

occurs

in the ATP

level. Further lation

work on the mechaniwne

of metabolic

regu-

of yeast and muscle PFKs ia in progress,

Aisenberg,

B.C.,

and Potter,

V.R.,

J. Biol.

Chem., 224,

1115 (1957).

Alberty, R.A., J. Am. Chem. Sot., 75, 1928 (1953). Chameux, J., Cold Spring Harbor S.ymp. on Quant. Biol., - 3,

3i3

(i961).

26,

277 (1961).

-

-

;erh.ert, J-C., and Pardee, A.B., J. Biol. Chem., 237, Ztn+ f*nfz.?\ Cold Spring Herbor Symp. on Quant. Biol., Holzer,‘H,, Lon'Fj;Eirg-Holm, ILK., Biochim. Biophys. Acta, 35, 464 (1959). Lynen, E., Hartmann, G., Wetter, K.F., and Schuegraf, A., in G.E.W. Wolstehholme and C.M. O'Connor (Editors) Regulation of Cell Metabolism, J. & A. Churchill, London, 1959, p. 256. Maitra, P.K., and-Steinberg, S.E., Federation Proc., 22, 240 (1963).

Mansour,

T.E., and Manaour, J.M., J. Biol. Chem., a, ( 1962). Yoyed, H.S., and Umberger, H.E.,Physiol. Revs., $2, 444 629

(1962).

Regelein, E., Biochem. Z., 287, 329 (1936). Rewsholme, E.A., and RandleT.J., Biochem.J., 80, 655 (1961). Park, C.R., Morgan, H.E., Henderson, Y.J., Regen, D.M., Cadenas, E., and Post, R.L., Recent Progr. Horm. Rea., IJ, 493 (1961). Passonneau, J.V., and Lowry, O.H., Biochem. Biophys. Res. commull., 2, 10 (1962). Vifiuela, E., and Sols, A., in preparation (1963). 145