Affinity labeling of AMP-ADP sites in heart phosphofructokinase by 5′-p-fluorosulfonylbenzoyl adenosine

Vol.

81,

April

28,1978

No.

4, 1978

BIOCHEMICAL

AND

LABtLING

OF

AMP-ADP

SITtS

BY 5’-p-FLUOROSULf-

Tag

2

RESEARCH

COMMUNICATIONS Pages

AFFINITY

1

BlOPHYSlCAL

Department

Mansourl

of Chemistry,

March

14,

PHOSPHOFRUCTOKINASL

ONYLBLNZOYL

and

of Pharmacology, Stanford,

Department

Received

t.

IN HtART

ADtNOSINt

Roberta

Stanford California

University

1370-1376

F. Colman2

University 94305

School

of Delaware,

of Medicine,

Newark,

Delaware

19711

1978

SUMMARY: The purine nucleotide derivative, 5’-p-fluorosulfonylbenzoyl adenosine (5’FSO B Ado) functions as an affinity label for the allosteric sites of phosphofructokinage . ’ The modified enzyme at pH 6.9 is insensitive to allosteric inhibition by ATP, activation by AMP, c-AMP, ADP and shows no sigmoidal kinetics for fructose-6-P. The reaction does not appear to occur at the catalytic site since modification of the enzyme does not significantly affect its specific activity nor its Michaelis constant at pH 8.2. ADP, and to a much lesser degree AMP and ATP, protects the enzyme from modification by the adenosine reagent. The modified enzyme essentially does not bind significant amounts of AMP, c-AMP, ADP, but still binds an analog of ATP, AppNHp. The adenosine affinity label will be of value in studies on the nature of the AMP-ADP allosteric sites.

Phosphofructokinase at neutral AMP

or

and

sites

the

through residue

However,

the

we

have

adenine

*

nature

binding found

that

(FSB-adenine)*

FSB-adenine

refers

the

from

of to

labels

a regulatory

site

adenine reacts

the

analog covalently

the

sites

and

allosteric

at pH

6.9

while

enzyme).

their

photooxidation

relationship

have to

provides

a

more

prior

covalent

reaction

0006-29lX/78/0814-137O$Ol.oo/o 1370

sheep

heart

been the

the made

catalytic of (Z-5).

effective

way

(6,7,8).

Recently, benzylthiol]

phosphofructokinase

benzylthio]

for

groups

8-[m-(m-fluorosulfonylbenzamido) with

and

modification

sulfhydryl

generally to its

sites

chemical

with

cyclic

inhibition

Attempts

(11,

reaction

Both

kinetics AMP,

of allosteric

sites.

and

or

8.2

presence

catalytic

to 8-[m-(m-fluorosulfonylbenzamido)

Copyright 0 I978 by Academic Press, Inc. All rights of reproduction in any form reserved.

at pH

enzyme

of the

anhydride

affinity

the

basis

by

ethoxyformic

the

“deinhibit”

allosteric

modification

by use

(or

on the

separate

kinetics

inhibits

inhibition

of

specific

histidine

selectively

are

order

ATP

explained

that

first

pH.

ATP

been

ligands

elucidate

acidic

relieve

have

modifier to

slightly

ADP

deinhibition

exhibits

adenine.

making

of

Vol. 81, No. 4, 1978

it

less

and

sensitive

ADP

AMP

inhibition

by

Modification

ADP

adenosine site

rabbit

muscle

modifier

of

binding.

A new

present

specific adenosine

**

bovine pyruvate of

investigation affinity reagents

in exploring

the

for exhibit

AMP-ADP

by this

shown

Since

whether

AMP-ADP

binding

(8)

sites chemical

the

and

of

is FSB-adenine,

that

reduction

of

reagent

label

of the

inhibitory

the

Mg-ADP

site

closely we

make

related have

Both both

of

to the

undertaken

functions

in phosphofructokinase. features

AMP

Y-p-fluorosulfonylbenzoyl

is more

adenosine

cyclic

a marked

as an affinity

5’-FS02BZAdo than

by AMP,

causes

derivative,

dehydrogenase

(IO).

different

reagent

to act

phosphofructokinase

the

to activation

nucleotide

been

to evaluate

label

insensitive

enzyme

glutamate

kinase

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and

purine

has

liver

nucleotides

ATP

of the

(5’-FS02BZAdo)

DPNH

the

to

(9).

and

BIOCHEMICAL

as a more adenine

of them

and

of value

sites.

METHODS: Sheep heart phosphofructokinase was purified and crystallized as described before (11, 12). Methods for solubilizing the crystalline enzyme and for assaying at pH 6.9 in imidazole buffer and at pH 8.2 in glycylglycine buffer were those described in previous investigations (9, 11, 12). The reagent 5’-FS02EZAdo was synthesized from adenosine and 5’-p-fluorosulfonylbenzoyl chloride by the method of Wyatt and Colman (10). It was dissolved in absolute ethanol 4and-lits lconcentration was determined spectrophotometrically at 259 nm (E= 1.35 x 10 cn M- ). For the determination of the C labelled sample of the reagent was amount of 5’-FSO B -adenosine bound to PFi&a prepared by the id&ion of 100 uCi of [8C] adenosine to nonlabeled adenosine (5 mmoles) prior to reaction with 5’-p-fluorosulfonylbenzoyl chloride. For a typical experiment to modify the enzyme and to measure the incorporation of the adenosine reagent, the enzyme (0.9 mg per ml) was incubated with the reagent at a final concentration of 50 uM for one hour in the presence of 100 uM of phosphate buffer pH 8.0 and 1 mM of EDTA. The concentration of ethanol in the reaction mixture was 4-6%. At the end of this period, the reaction was stopped by adding concentrated mercaptoethanol to bring the final concentration to 44mM. A control sample of the enzyme was treated exactly the same way without adding the adenosine reagent. In order to measure the amount of the reagent that binds covalently to the enzyme, the modified enzyme was dialyzed twice against 500 volumes of a solution containing 50 mM sodium phosphate buffer at pH 7.2 and 0.1% SDS for one hour and overnight against For preparation of a stock batch of modified 3000 volumes of the same dialysis buffer. enzyme, 5.4 mg of modified enzyme in 6 ml of buffer solution was precipitated by adding powdered ammonium sulfate (0.38 gm per ml) immediately following enzyme modification by the reagent. The precipitate was centrifuged at 12000 xg for 10 min and the residue was suspended in 1 ml of 50 mM phosphate buffer (pH 8.0) containing 100 uM ATP, 10 uM fructose-1,6-P2, 1 mM EDTA and 1 mM dithiothreitol. To this enzyme solution was added, slowly, an equal volume of 100% room temperaturesaturated ammonium sulfate at pH 7.8 and O”, with constant stirring. The suspension was stored at 4OC overnight. The enzyme was then centrifuged at 7000 xg for 10 min. The residue was suspended in 50 mM phosphate buffer (pH 8.0) containing 100 uM ATP, 10 uM fructose-1,6-P , 50 mM DTT and 50% saturated ammonium sulfate. The enzyme can be stored in this i! orm for months.

**

5’-FS02BZAdo

refers

to 5’-p-fluorosulfonylbenzoyl

1371

adenosine.

Vol. 81, No. 4, 1978

RESULTS

AND

reaction

DISCUSSION:

of the

from

0.96

BIOCHEMICAL

enzyme

to

1.16

of

reagent

was

not

protein

was

indicated

solution

the

did

the

6.9.

of

however,

hand,

ATP

AMP.

that

the

is

that

the

native

the

modified

curves,

however,

shown).

Modified

enzyme

investigated

App(NH)p

ADP

against

being

caused

almost

protect

the

enzyme

SDS

the

dialysis

marked

difference

than

to activation

by

enzyme

and

from

ATP

modification

inhibition.

against

1372

modification

At and

with results

at concentrations activiation.

For

fructose-6-phosphate

the

pH

caused ADP

caused

by the

reagent

Fructose-6-phosphate, reagent.

ATP.

(data

not

pH (6.5).

reactivity

8.2

AMP

of

kinetics

AMP

by the

other

AMP The

at an acidic on

0.5

of 1 mM

concentrations

protection. while

AMP

no sigmoidal

native

ATP

enzyme.

the

inhibitory

6.9,

of

of

the

or presence

to AMP on

show

On

effect

native

presence

up to a level

the

at

becomes

In the

enzyme.

the

examined

enzyme

absence

nucleotides

at pH

was

uM.

native

velocity

complete

to ATP

to

constants,

inhibition

insensitive

different

modification

desensitized

of

enzyme

that

enzyme

the

Michaelis

modified

illustrate

of

stable of

I shows

the

2

presence

more

maximum with

the

no

as 700

in the

initial

modified

Table

of

the

inhibition

of

influence

chemical

Protection

did not

is also

inhibits

is completely

the

of the

the

5’-FSD2BZAdo.

from

in

ATP

is sensitive

dependence

varied

bound

inhibition

that

from

enzyme

60%

showed

as high

Fig.

enzyme

sigmoidal

Titration

while

cause

covalently against

to ATP

1 show

ATP

enzyme

the

Fig.

is protected

in

ATP

enzyme

concentrations

summarized

concentration

enzyme

at

8.2,

The

incubation

“Methods”).

pH

that

enzyme.

and modified in

of

was

dialysis

under at

(13)

modified

enzyme,

protection.

native

reagent

hr

after

a value

= 90,000).

one

exhaustive

plots

enzyme

inhibit

the

gave

(MW

following

(see

the

while

protection

reagent

ATP

native

of

protection

the

not

native

with

that

summarized

results

8.2

of radioactivity

by dialysis

protomer

That

concentrations

as 10 uM

We

pH

at higher does

concentrations

fact

native

to

the

per

at

the

the

both

results

The

as low

and

of

AMP

mM,

show

by

insensitive

1 mM

reagent

Lineweaver-Burk

The

completely

the

incorporation

followed

changed.

release

Sensitivity pH

of

measured

modified

of the

5’-FS02BZAdo

significantly

from

between

with

enzyme

not

determined

Measurement

mole

velocity

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of only caused only

PFK

partial good partial

prevents

the

citrate,

and

BIOCHEMICAL

Vol. 81, No. 4, 1978

AND 8IOi’HYSlCAL

RESEARCH COMMUNICATIONS

0.7 0.6 t

2 z

0.5-

8 d a4a. .z -0 0.33 0.2 0.1 01

0

I 0.1

I

I

I

I

I

0.2

0.3 [ATPI

0.4

0.S

0.6

I

0.7

mt.4

I 0.2

02

I 0.4

1 0.6

to inhibition by were prepared as was measured at 6.9 without AMP fructose-6-P was

Fig. 2. Sensitivity of native (a) and modified 0) phosphofructokinase to activation AMP. Enzyme activity was measured at pH 6.9 in the presence of 0.25 mM ATP, mM fructose-6-P and 1 mM MgC12

Protection Incorporation Nucleotide

5FS02BZAdo

Incorporated

by 0.5

I

by Nucleotides

of [14C]-5’-FS02BZAdo

I I.0

[AMPI mM

Fig. 1. Sensitivity of native (A) and modified 0 phosphofructokinase ATP. Enzyme modified by 5-FSBO B -adenosine and native enzyme described under “Methods .” Imti* *a? ve4 ocity of phosphofructokinase different ATP concentrations in the regular reaction mixture at pH (straight line) or with 1 mM AMP (dashed lines). The concentration of fixed at 0.5 mM and the MgC12 at I mM.

TABLE

I ae

Against by Phosphofructokinasea

(Mole/mole

Phosphofructokinase)

pH 6.9

pH 8.0 None

0.97

1.01

5mM ATP

0.83

0.49

5mM ADP 5mM AMP

0.21 0.71

0.17 0.49

‘Reaction of the enzyme with the adenosine reagent was carried out for I hour as described under Methods except that the various nucleotides were added to the reaction mixture as indicated. The buffers used were 100 mM of potassium phosphate at pH 8.0, or pH 6.9 with 1 mM EDTA.

1373

Vol. 81, No. 4, 1978

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

r

h

\E E I 15

\

0.8

0.7

Q6

0;5-

a4-

0.3 -

0.2 o.l-s, - 0

0

04

0

02

The Figs.

of

3, 4 and

modified AMP not

effect

5 show

enzyme. and

0.9

bind

mole

to

modified

enzyme

do

imidoanalog, modified

of

The

differ

results

was of

is functioning

per

is

mole

not

the

0.s

1.0

the

shown).

one

binding

to

The class In

to the

native

Affinity

enzyme

binding

of

of sites

the

5).

as to the

0.65

to Binding

binding for

mole

native of ADP

and of

App(NH)p

native an by

low. the

present as

investigation

an affinity

label

1374

suggest

for the

that

allosteric

5’-p-fluorosulfonylbenzoyl sites

of does

the

modified

of

binding

examined.

essentially ADP

(Fig.

amount of

was

binds

cto

binding

as well

enzyme

contrast,

maximal

PFK

AMP native

to

0 phosphofructokinase. value for c-AMP

Modified

nucleotide.

in

06

phosphofructokinase, for AMP binding

native

enzyme.

abolished.

significantly

ADP

that

more than

almost

(data

c-AMP,

show

either

indicates

and

App(NH)p enzyme

adenosine

amounts

enzyme

not

plots

AMP

(0) value

on nucleotide

of AMP,

Scatchard

of cyclic

sigificant

is not linear

binding

0.4

(A) and modified 5 to 50 PM. KD

modification

the

The

enzyme the

enzyme

w\,

Moles Cyclic AMP Bound per Mole Enzyme

Fig. 3. Binding of AMP to native (4) and modified concentrations were varied from 5 to 50 uM. K,, enzyme was 4.87 MM. Fig. 4. Binding of cyclic AMP to native AMP concentrations were varied from native enzyme was 0.72 uM.

,

of phosphofructo-

ATP the

Vol. 81, No. 4, 1978

BIOCHEMICAL

Moles

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

0.6 ADP Bound

1.2

0.6 1.0 per Mole Enzyme

1.4

ADP

Fig. 5. Binding of ADP to native (Is) and modified (0) phosphofructokinase. concentrations were varied from 4.5 to 112.8 uM. K value for ADP high of native enzyme was 0.82 pM and 12.4 pM for low a9. fimty * sites.

kinase. the

Reaction enzyme

supports

does the

is different are by

similar the

does

from

catalytic

appreciably

bind

inhibition

by

allosteric

site.

initial

capable

enzyme protection

by

or

its

c-AMP:

on

ADP.

possibility

provide

inhibitory

analog)

not

of the

of the effect

at a distinct

that

modified

inhibitor, marked

1375

and

two

by

types

the

even

if

the

cannot

the

enzyme addition

reaction

provide

exclude

of of the

appreciable sites seem

might

to

inhibitory

altered

of allosteric

not

it

the

deinhibitors)

it does

is occupied

is insensitive

since

by 5’-FS02BZAdo

enzyme

and

does

ADP

a hyperbolic

against

ADP

result

and

site

within

protection

The

site

site.

AMP

exhibits

since

of

This

modified

enzyme

ATP,

dehibitor

occupied

ATP

for

allosteric

modification

reagent.

of added

site

to be excluded

deinhibitor

8.2.

concentration the

the

pH

enzyme

sites

modification

of the

the

modified

the

In contrast,

and

allosteric

in which

seems

since at

properties

a direct

does

inhibitor

the

the

Since

postulate

incorporation

activity

that

the

of

mixture

site

fructose-6-phosphate

or

might

catalytic

specific

enzyme

an analogue

the

the

native

ADP

the

Moreover,

of binding

Occupation

eliminate (or

for

5’-FS02BZAdo.

distinguishable.

of ATP

(1,2)

this

against

occupied

previously

However,

reaction

with

at its

AMP

one

occur

significantly

velocity

c-AMP,

ATP,

to

site.

expected AMP,

of

to

the

to those

to the

affect reported

deinhibitors

is still

appear

evidence

dependence

ATP

not

not

affinity

(that to

be expected the

binding

be

Vol. 81, No. 4, 1978

BIOCHEMICAL

The present better

affinity

tested

previously

AMP

reagent,

label (9).

addition

to

The extent

orientation

the

affinity

sites

following

ribose

label

than

moiety

will

its analog

reagent must

moiety be of value

may be considered FSB-adenine

to ATP inhibition

modification

the adenine

RESEARCH COMMUNICATIONS

adenosine,

of desensitization

of the fluorosulfonylbenzoyl

and the adenosine ADP allosteric

following

adenine,

5’-fluorosulfonylbenzoyl

for the AMP-ADP

and ADP was complete

was less extensive

AND BIOPHYSICAL

by the adenosine (9). play

It therefore

an important

of the affinity in studies

label.

a

which

was

and activation

by

reagent

while

it

appears

that

in

role

for

optimal

Both the adenine

on the nature

of the AMP-

sites.

ACKNOWLEDGEMENT: We wish to thank Miss Linda Lan for her excellent technical assistance. This investigation was supported by U.S.P.H.S. Grant HL17976 and American Cancer Society Grant B.C.-138. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Ahlfors, C.E., and Mansour, T.E. (1969), J. Biol. Chem. 244, 1247-1251. Setlow, B., and Mansour, T.E. (19701, J. Biol. Chem. *724-5533. Kemp, R.G. (19691, Biochemistry g, 4&91-4496. Younathan, E.S., Paetkau, V., and Lardy, H.A. (19681, J. Biol. Chem. 243, 1603-1608. Froede, H.C., Geraci, G., and Mansour, T.E. (19681, J. Biol. Chem. 243, 6021-6029. Anderson, R.A., and Graves, D.J. (1973), Biochemistr 12, 1895-1906. 2, Anderson, R.A., Parrish, R.F., and Graves,-$,8’iochemistry 1901-1906. Paf, P.K., Wechter, W.J., and Colman, R.F. (1975),-J. 3iol. Chem. 250, 8140-8147. Mansour, T.E. and Martensen, T.M. (19781, J. Biol. Chem. (In Press). Wyatt, J.L. and Colman, R.F. (19771, Biochemistr 16 1333-1342. Mansour, T.E., Wakid, N., and Sprouse, ---?-+-’ H.M. 1966, J. Biol. Chem. 3, 1512-1521. Lorenson, M.Y., and Mansour, T.E. (1969), J. Biol. Chem. 244, 6420-6431. Mansour, T.E. and Ahlfors, C.E. (19681, J. Biol. Chem.. 243, 2523-2533.

1376