Binding of nucleotides AMP and ATP to yeast phosphofructokinase: Evidence for distinct catalytic and regulatory subunits

Vol. 80, No. 3, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

BIOCHEMICAL

Pages

February 14, 1978

646-652

BINDING OF NUCLEOTIDES AMP AND ATP TO YEAST PHOSPHOFRUCTOKINASE:EVIDENCE DISTINCT

Michel

LAURENT, Alain

CATALYTIC

FOR

AND REGULATORY SUBUNITS

F. CHAFFOTTE, Jean-Pierre TENU, Colette FranGois J. SEYDOUX.

ROUCOUS and

Laboratoire d'Enzymologie Physico-Chimique et Moleculaire Universitk de Paris-Sud, 91405, Orsay-France.

Received

October

27,

1977

SUMMARY The binding of ATP to yeast phosphofructokinase, as monitored by flow dialysis, is heterogeneous and is adequately described by assuming two independent classes of three binding sites each per enzyme molecule. Under similar conditions, theming of 5'AMP is homogeneous and a binding stoichiometry of three S'AMP molecules per enzyme molecule is evaluated. Displacement experiments show that only the ATP molecules bound to the first class of three tight binding sites are displaced by an excess of 5'AMP. Thus, these tight ATP binding sites can be identified as "regulatory sites" in agreement with kinetic data. Furthermore, molecular weight determinations and electrophoresis results are consistent with an heterologous a 6, structure of the enzyme oligomer. Therefore the present binding data sugges ? that yeast phosphofructokinase is constituted by three "catalytic" and three "regulatory" subunits. .

INTRODUCTION Phosphofructokinase pathway, tic

shows a great

organisms.

number

In the

of subunits

now well

of two types

weight

(4).

are

easily

degradation features

These

variability case of yeast that

the

two types

in the presence of the yeast

a matter

native

form

are

relative

of specific

enzyme are

not unique

646

in their

immunologically (5).

the exact

(1,2,3),

enzyme contains slightly

it

is

an equal molecular

distinct

susceptibility substrates

0006-291X/78/0803-0646$01,00/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

of this

and eukaryo-

although

of controversy

c1 and 6 differing

of subunits by their

among prokaryotic

phosphofructokinase,

still

of subunits

differentiated

a key enzyme of the glycolytic

of structures

(6 or 8) is

established

number

(EC 2.7.1.11),

(4)

and

to proteolytic These

among eukaryotic

structural phosphofructokinases

BIOCHEMICAL

Vol. 80, No. 3, 1978

since

the enzyme

contain

two types

weight

quaternary

significance

from human erythrocytes of subunits structures.

has been given

phosphofructokinase. that

yeast

(i.e

"catalytic"

binds sites)

(i.e

equilibrium

binding data

be correlated

are

however,

contains

data

for

with

no particular (8)

class

of sites

The allosteric

the occurence leading

our previous

binds

of two equally

distributed that

catalytic

in yeast showing sites;

reaction

ATP as an allosteric 5'AMP modifies work,

we report

phosphofructokinase.

kinetic

to the conclusion

of distinct

molecular

of ATP binding

enzymatic

In the present

ATP and 5'AMP to yeast with

data

activator

sites.

high functional

kinetic

in the

to also

of subunits

classes

donnor

(7)

complex,

of two types

two distinct

other

sites).

found

into

presented

ATP as phosphate

in agreement

to be composed

MATERIAL

now,

to the regulatory

in the enzyme oligomer, likely

Until

and the

of ATP only

is

distributed

to the occurence

"regulatory"

the binding binding

are

We have previously

of sites

inhibitor

has been recently

which

phosphofructokinase

one class

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

These

measurements types yeast

and regulatory

and can of subunits

phosphofructokinase subunits.

AND METHODS

Phosphofructokinase was prepared from baker's yeast according to the slightly modified procedure of Diezel et al. (1). Unlabelled 5'AMP and ATP were purchased from Sigma. Radioactive [Ul"C] ATP (562 mCi/mmol) and (U14C] 5'AMP (538 mCi/mmol) were obtained from the Radiochemical Center Amersham (England). All other chemicals were of the best available purity. The evaluation of ATP and 5'AMP binding by gel-filtration experiments was performed as previously described (9). Flow dialysis (10) measurements were conducted according to Tenu et al. (11) using Instagel (Packard, France) as scintillation liquid. All binding experiments were carried out at 25OC, pH 6.8, in 50 mM Tris-sulfonate buffer containing 25 mM K2HP04, 1 mM dithiothreitol, 5 mM MgC12 and 1 mM EDTA. Phosphofructokinase concentrations were determined from refraction index measurements, assuming an increment value dn/dc of 1.85 (mg/ml)-l ; this value leads to an absorption coefficient of 0.97 A/mg at 280 nm. Cross-linked hemoglobin and bovin serumalbumin prepared according to the procedure of Payne (12) were a generous gift of Dr. A. d'Albis. The electrophoresis technique was adapted from that of Taucher et al. (6) using a 4% polyacrylamide gel. Light scattering measurements were performed as described by Dessen and Pantaloni (13). RESULTS QUATERNARY STRUCTURE OF YEAST PHOSPHOFRUCTOKINASE. The molecular evaluated

by light

scattering

730 000 + 30 000 daltons by Diezel Hess (2) sodium

et al.

weight

(1)

dodecylsulfate

phosphofructokinase

and gel filtration 1). This value

(fig.

and agrees

in the case of the

of native

very

well

brewer

yeast

polyacrylamide

gel

647

has been consistently

on Utrogel AcA 22 (LKB) to is slightly smaller than that reported

with enzyme.

that

reported

by Tamaki

and

in fig.

2,

As illustrated

electrophoresis

performed

on freshly

BIOCHEMICAL

Vol. 80, No. 3, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I Q7 9’ -f

5

01

CONCENTRATION

02

IfTMJ/fllll

Fig.

1 :

scattering data for yeast phosphofructokinase. Plot of the relative scattered intensity at 436 nm, 20°C, versus protein concentration as obtained for two distinct enzyme preparations ( 0 and 0). Scattered intensities are extrapolated to 0" angle and are normalized with respect to the scattered intensity of the reference glass of the FICA 50 photogoniometer.The least square fitted straight line has a slope of 10.84 + 0.39 intensity units/mg of protein. From this value, a molecilar weight of 730 000 ;t 30 000 daltons can be calculated for yeast phosphofructo kinase, using a calibration factor of 67 000 + 400 daltons per unit of scattered intensity/me of protein (13f.Note that no significant departure from linearity is observed, as an indication of a negligible contribution of the second virial coefficient.

Fig.

2 :

Polyacrylamide gel electrophoresis of yeast phosphofructokinase in 0.1% sodium dodecyl sulfate. Experimental conditions : 4% acrylamide gels , 5 hrs. of migration at 8 mA/gel tube, 20 ng of protein were applied. Staining of the bands was achieved with Coomassie brilliant blue (6). According to the densitometric analysis shown on the right,the a, B, a' and B' bands are in the ratio 1 : 0.87 : 0.22 : 0.18 respectively. The amount of proteolysed material (IX' and B' chains) was particularly high in this one week old enzyme preparation. For most enzyme preparations used in the present work, when electrophoresis was carried out immediately after the last purification step, the partially proteolysed material amounted to 5-10" o of the total protein material.

prepared

Light

enzyme shows the

et al. (1). A strong followed by a rather a' and B' chains

typical

migration

doublet corresponding faint doublet which

(about

5-10% of the

weight of these subunits with a series gives the values of 124 000, 113 000, a' and B' chains,

respectively.

pattern is

total

previously

reported

to the native a and B chains is formed by the partially proteolysed protein).

Evaluation

of cross-linked proteins 95 000 and 88 000 daltons

These values

648

by Diezel

were

confirmed

of the

molecular

as markers for the cx, 6,

by electrophoresis

Vol. 80, No. 3, 1978

with

BIOCHEMICAL

uncrosslinked

filtration

molecular

experiments

results,

native

by the

association

F. Seydoux

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

weight

markers

on Sepharose

yeast

appears

a and three

and J. Yon, manuscript

dodecylsulfate

and by gel

CL 6B in 6 M guanidine.Thus,

phosphofructokinase

of three

in sodium

from

as an hexameric

8 chains

(i.e

these

enzyme formed

a3B3 structure,

A. Chaffotte,

in preparation).

BINDING OF !5'AMP AND ATP TO YEAST PHOSPHOFRUCTOKINASE. The binding monophasic,

isotherm

and a stoichiometry

determined

viith

binding

constant of

data

per

is clearly

oligomer

the binding

biphasic,

can be fitted

3a,

can be

16 nM.

S'AMP,

is distinctly

the binding

sites

of about

to the binding

phosphofructokinase

Accordingly,

as shown in fig.

of three

a dissociation

In contrast to yeast

of 5'AMP,

isotherm

of ATP

as shown in fig.

to the

following

3b.

equation

:

n(ATPjfree ATP bound which

accounts

sites.

for

of sites

constants

loose

binding

Thus,

yeast

three

binding

enzyme (mol/mol)

the binding

The number

dissociation

per

are

in the

sites

by flow

experiments

using

stoichiome;:ries,

it

sites

regulatory only

by an excess that

5'AMP rapidly

behavior hexamer. binding catalytic the

Thus, sites

the this

of sites

similar

and

of

bound

allows with (36.5

kinetically

of our

us to identify

this

second

class

interpretation,

uM) compares for

conditions. 649

well

the catalytic

be

dialysis

ATP was displaced experiment

shows

an appreciable

5'AMP concentrations.

by assuming

that

only

kinetic

should

4, this

that

binding

previous

in a flow

in fig. ATP but

filtration contains

sites

of bound

to the three

and the

stoichiometries by gel

From these

tested

amount

bound

sites

agreement

ATP (31 FM) as determined

In view

has been

described

experiment

binding confirmed

to the regulatory

As depicted

ATP molecules

In

classes

phosphofructokinase

enzyme even at high

class

two distinct

peculiar

yeast

This

tightly

the

as regulatory

sites. second

that

bound

5'AMP.

can be quantitatively

more efficiently

nM respectively).

to contain

of

and the

2.3 and 36.5

substoichiometric

displaces

boundto

to three

of 15 (i.e

enzyme preparations.

by S'AMP.

small

of unlabelled

of ATP remains

for

a

close

number

to the tight

per enzyme oligomer.

ATP molecules

where

of equal

respectively

1, these

different

displaced

experiment

was found

5'AMP and ATP were

can be deduced

the

significantly

with

several

classes

K2 + (Wfree

ATP.

in Table

dialysis

(n)

+

correspond

appears

each for

three results,

ratio

phosphofructokinase As illustrated

obtained

in each class

n(ATP)free

Kl + (A'Wfree

of ATP to two distinct

and K2 which

K1

sites,

= ; =

fraction This

5'AMP displaces

tight

sites

the first of sites the with

of the class

enzyme

of ATP

as the

dissociation the

reaction

constant

Km value (8)

for

under

Vol. 80, No. 3, 1978

l-i g.3

:

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Binding isotherm of 5'AMP and ATP to yeast phosphofructokinase as determined in flow dialysis experiments. Scatchard plot of the data ; corresponds to the apparent number of bound ligand molecules per enzyme oligomer. (a) Binding isotherm of 5'AMP. c]: 5.2 UM enzyme. a : 12.6 PM enzyme The solid line is calculated for a single class of 2.75 sites per enzyme oligomer with a dissociation constant of 16.2 FIM. (b) Binding isotherm of ATP : 3.9 uM enzyme. Solid line is calculated fromequation 1 with Kl q 2.35 uM, K2 q 36.6 PM and n = 3.07.

1.5

50 [5’AMP] Fig.

4 :

pM

Displacement of ATP by 5'AMP from yeast phosphofructokinase as measured by flow dialysis. Experimental conditions : 4 PM enzyme, 1.93V M (U-14C)ATP. The solid curve is calculated by assuming a competitive displacement of ATP by 5'AMP from the regulatory tight binding sites as discussed in the text. The corresponding equation used in that case is obtained by multiplying the dissociation constant K in equation 1 by (1 + AMP/K), K (=9 PM) being the dissociation coAstant for 5'AMP ; other parameters values as given in fig. 3b.

650

BIOCHEMICAL

Vol. 80, No. 3, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE 1 Comparison of the data obtained by gel filtration and flow dialysis the binding of ATP and 5'AMP to yeast phosphofructokinase. Apparent numbers of bound ligand molecules per enzyme oligomer (;) measured in gel filtration experiments were obtained with 3 distinct enzyme preparations. ( ( ( ( ( ( i

Ligand

.:

Concentration'=) ( PM)

for

;

i : gel

filtration

: flow

dialysis (b)

(

ATP

:

ATP

i

9.30 47.5

: 3.02

t

0.37

:

3.05

: 4.95

+

0.40

:

4.62

) ) ) ) ; 1 ) i

i ( (

5'AMP

(a) free

:

ligand

(b) These

69.3

: 2.45

t

0.20

:

2.23

; 1

concentration.

values

were

interpolated

from the

data

of fig.

3.

DISCUSSION Although structure,

the

the present

essential

yeast

phosphofructokinase

sites

which

oligomer.

is

equal

contains

to only

in view

to assume that

the

lytic for

half

"303

ATP as phosphate

donnor.

In this

of asymmetrical

by our previous fructokinase

kinetic with

of three

interacting

pretation

should

and functional

of regulatory

and catalytic structure

ATP or 5'AMP and each catalytic

as a trimer

an hexameric work

sites,

of yeast

dimeric results

respect

but

per

that

6-phosphate

be substantiated by additional yeast phosphofructokinase

levels,

651

interpretation

the cooperativity

enzyme oligomer

that

that the enzyme one type

the most plausible

phosphofructokinase

contains only enzyme oligomer This

c1 fi units.

showing

to fructose

protomers

subunit sense, the

"3B3 is

ATP and 5'AMP binding

the number of subunits constituting we cannot exclude the possibility

regulatory

of the

with

of the present

enzymeoligomer is composed of three regulatory each regulatory subunit should contain Therefore,

subunits. for

a number

data,

both

interpretation

site

are consistent conclusion

contains

From the present

of subunits

data

qualitative

is

and three cataone regulatory one binding site should be considered is supported of yeast

phospho-

was best explained on the basis this tentative inter (8). Although evidence at both structural may constitute a new example

BIOCHEMICAL

Vol. 80, No. 3, 1978

of a highly distinct mylase

specialized subunits,

from

E. coli

allosteric

as already

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

enzyme with described

for

the

structurally

and functionally

enzyme aspartate

transcarba-

(14).

ACKNOWLEDGMENTS We are indebted to Professor J. Yon for her interest to this work. We are very grateful to Dr. A. d'Albis for her generous gift of cross-linked proteins and to Dr. Pantaloni for his assistance and advices concerning light scattering measurements. We acknowledge Dr. Thusius and M.J. Lavorel for careful reading of this manuscript. This work was supported by grants from the Centre National de la Recherche Scientifique (G.R. no 13).

REFERENCES

(1)

(2) (3) (4) (5)

(6) (7) (8) (9) (10) (11)

(12) (13) (14)

Diezel, W., Bdhme, H.J., Nissler, K., Freyer, R., Heilman, W., Kopperschlzger, G. and Hofmann, E. (1973) Eur. J. Biochem. 38, 479-488. Tamaki, N. and Hess, B. (1975) Hoppe Seyler's Z. Physiol. Chem. 356, 4, 399-415. Kopperschlgger, G., Usbeck, E. and Hofmann, E. (1976) Biochem. Biophys. Res. Commun. 71, 371-378. Herrmann, K., Diezel, W., Kopperschlgger, G. and Hofmann, E. (1973) FEBS Lett. 36, 190-192. Huse, K., Kopperschlgger, G. and Hofmann, E. (1976) Biochem. J. 155, 721-723. Taucher, M., Kopperschlager, G. and Hofmann, E. (1975) Eur. J. Biochem. 59, 319-325. Karadsheh, N.S., Uyeda, K. and Oliver, R.M. (1977) J. Biol. Chem. 252, 3515-3524. Laurent, M. and Seydoux, F. (1977) Biochem. Biophys. Res. Commun., 78, 1289-1295. Seydoux, F., Kelemen, N., Kellershohn, N. and Raucous, C. (1976). Eur. J. Biochem. 64, 481-489. Colowick, S.P. and Womack, F.C. (1969) J. Biol. Chem. 244, 774-717. Tenu, J.P., Ghelis, C., Yon, J. and Chevallier, J. (1976) Biochimie 58, 513-519. Payne, J.W. (1973) Biochem. J. 135, 867-873. Dessen, P. and Pantaloni, D. (1973) Eur. J. Biochem. 39, 157-169. Gerhart, J.C. and Schachman, H.K. (1965) Biochemistry 4, 1054-1062.

652