The inhibition of pyruvate kinase by ATP

Vol.

3 1, No.

BIOCHEMICAL

5, 1968

THE

AND

INHIBITION

OF

PYRUVATE

T. Department

Received

of Biochemistry,

(Meyerhof

of authors

and

Cohn,

Oesper,

1966;

incorporated 1964;

into

appeared

schemes and

these

action

of ATP

on the

of the

experimental

revealed

that

ADP

PEP for

and

found

In the by ATP

by Reynard

depends

Quebec

level

ATP

by Reynard

et al.

kinase, and

can Utter

the

effects

inhibitory

ATP

to competion

be accounted (1959)

the

A careful reported

(1961)

Passoneau,

laboratory

to reduce

+ pyruvate). have

and

in this

eliminated

who

Mildvan

has been

(Lowry

out

designed

workers

raised

to a point

metal,

and

Methods kinase reaction Initially,

the

activity with the

are

reported

of magnesium

was

Materials

63).

carried

by ATP

1961;

inhibition

in brain

completely -

kinase

et al.,

of this

experiments

by various

experiments

was partially

the

coupled

p,aper,

(1961)

with

Pyruvate

Reynard

experiments

changes,

McQuate

pyruvate

of glycolysis

further

of pyruvate

In fact,

1959;

occurrence

control

attributed site

the

(PEP + ADP

used

of muscle

Utter,

Some

of pH

reaction

on the

saturated

Angeles,

Montreal,

for

explained

scrutiny inhibition with

by its magthe

inhibition

basis,

present

concentration

in the

forward

action.

et al.

but

and

inhibition,

on this

the

findings

the active

chelating

and

1967).

conditions

this

and

1967)

Newsholme,

to confirm

inhibition

McQuate

to explain

of magnesium

tion

BY ATP

University,

reported

et - al,,

of complexing

they

KINASE

COMMUNICATIONS

Wood

McGill

have

1949;

Tanaka

Rolleston

nesium

RESEARCH

May 3, 1968

A number

and

BIOPHYSICAL

in the

reproduced. where

inhibition

all

conditions

demonstrate

that

medium,

and

inhibition

However,

the

when

magnesium-complexing

the the

the

inhibiobserved

magnesium species

were

disappeared.

was measured lactate

which

by following

dehydrogenase approximated

779

the

rate

in a Calbiometer those

described

of NADH

oxidation

(Calbiochem, by Czok

Los and

Vol.

3 1, No.

5, 1968

Eckert

(1965)

muscle

pyruvate

(360 and

but were

dilution

(125

5 mg/ml) diluted

of

obtained

lactate

RESEARCH

as shown

2 mg/ml) from

use in 1 mg/ml

and

BIOPHYSICAL

modified

I. U./mg,

were

before

1 : 400

AND

progressively

kinase

l.U./mg, were

8lOCHEMlCAL

in Table

and

bovine

Crystalline

lactate

Mannheim

serum

Pyruvate found

rabbit

dehydrogenase

Corp.,

albumin.

at 1 : 10 were

dehydrogenase

1.

crystalline

boehringer

COMMUNICATIONS

New

York,

kinase

at a

to be stable

for

at

east

6hrsatO’. In the amounts was

of KCI

added

the

Diluted had

Readings

MgCi2

ADP,

PEP,

the slope

(if

the

of the

ml of a buffer

experiments

present),

was

linear

portion

of the

the

pH of the

cuvette

added

water added

to a volume and

at 30 second

time

and

the

plot

which

when

of pyruvate for

velocity

end

to this ml.

the op+ical

kinase.

run

minutes.

calculated

beyond

of the

density

lo-14

was

extended

at the

required

of 2.00

intervals

initial usually

contents

the

separately),

by the addition

taken

against

containing

was

and

started

mp were

plotted

MgC12

NADH,

reaction

at 340

solution

ml of 1 : 10) was

(0.01

were

experiments,

6 minutes.

was

checked

a pH-meter.

Results

listed

and

Discussion

The

initial

in Table

convert

Group

velocities

1 as optical

by ATP,

of enzyme

was

be detected.

when

increased

was

was when

raised, EDTA

of the

was

inhibition disodium

low

the and

under

of the

(a)

for

absence much

greater

EDTA

was

absent.

inhibition

In group observed

by 3.11

even

by EDTA.

when

the

amount

no activity

by ATP (d),

when but

to

expected

complexed

magnesium

inhibition

are

(divide

the

magnesium,

was

conditions

enzyme

is already

of extra

very

reaction

demonstrate

magnesium

requirement

of ATP

various undiluted

in group

in the

a lack

the

could

when the

the

mag-

magnesium

inhibition

was added. made

obtained salt

results part

400-fold

kinase

units/minute/ml

absolute

(c) shows

concentration

An attempt the

a large the

Group

concentration reappeared

density The

(b) demonstrates

nesium

of pyruvate

to pmoles/min./ml).

inhibition

but

ATP

density

densities

1 .50

( in a few

value

of optical

In some

placed

dehydrogenase a steady

The optical

with

was

and

lactate

reached

from

cuvette

of ATP

to reproduce (group which

(e))

the was

reduced

found the

780

inhibition

observed

subsequently pH of the

reaction

by

Reynard

to be due mixture

to the

et al .(1961) low

considerably

acidity at

Vol.

3 1, No.

the

low

BIOCHEMICAL

5, 1968

buffer

concentration

used,

AND

When

the

Table Inhibition

BIOPHYSICAL

pH of the

RESEARCH

ATP

COMMUNICATIONS

was adjusted

to 7.0,

the

I

of Pvruvate

Kinase

bv ATP Rate ODu/min./ml

(a)

(b)

46 mM 7.3

triethanolamine mM MgC!2,

50 mM 1 .l

imidazole mM MgC,‘2,

as above

using

(c)

50 mM

imidazole

(d)

50 mM

imidazole

buffer, 8.9 mM pH 7.6 (1) +SmMATP buffer, 8.9 pH 7.5

0.01 II II

ml

mM

TO mM

glycylglycine

(f)

20 mM

glycylglycine

as above,

pH

67 mM

KCI, 740 90

EDTA,

67 mM

KCI, 0

+ 10 mM undiluted enzyme

MgCl2

635 0

buffer,

12 mM

KCI, 1 .l +5mMATP

buffer,

67 mM

KCI, 7.5 mM MgC12, + 5 mM ATP + 5 mM ATP + 10.3 mM

8, (e)

EDTA,

I,

7:;

mM

MgC12,

pH

7.5

184 6

pH 7.5

480 490 20

EDTA

buffer,

100 mM KCI, + 5 mM ATP

10 mM (2)

MgC12,

pH 8.5

328 58

buffer,

100 mM KCI, + 5 mM ATP ATP (3)

10 mM (3)

MgC12,

pH

367 340 405

+ 5 mM

8.5

All experiments were carried out at 30°, 0.25 mM ADP and 0.37 mM addition of 0.01 ml of pyruvate kinase diluted 1 - 400 with 1 mg/ml (1) Conditions of Czok and Eckert (1965); (2) pH of solution was 3.6; was 7.0. inhibition tion

was

slight

observed

both

magnesium

left

for

complexing

tion

and,

as shown

when

the

magnesium

ratio

rose

above

Published

Melchior

kinase

action

8.5.

by the

which

The

(1961)

and

uncomplexed

pyruvate

I n a further

et al.

at pH 7.5

to Mg-ADP,

substrate

(f)).

by Reynard

reproduced

present

(group

of ATP

1 the

concentration until

values

the order

of 2 x lo3

Melchior

(1965)

and

the

2 x lo4

has pointed

a 2 mM

added

ATP

out

has produced

was and

magnesiun

amount

to convert

all

the

to show

produces

inhibition

disappeared

no inhibition

could

presence

ADP1965;

of 200

mM

a fall

concentra-

the

be detected and

Mg

Mildvan potassium

greatest Mg

: ATP

at all.

ATP2’ and

true

in rate.

was when

of

ADP

is the

magnesium

gradually

(Melchior,

781

the

The

of Mg

inhibi-

of ADP,

the

reversed.

constants

respectively in the

of ATP

2) the

quantitatively,

evidence

by increasing

inhibition low

not

is insufficient

addition

formation

that

but

(Table

concentration

be overcome

was

at 15 mM for

At

(1960)

can

of experiments

was qualitatively,

- consequently

in Figure

1,

series

PEP with the bovine serum albumin. (3) pH of solution

are Cohn,

the greater

of 1966).

Vol.

3 1, No.

5, 1968

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

V O/V

5

16

12

8

4

4

8

12

Magnesium

Figure

1.

inhibition af added

of the

amount necessary. nesium

ATP

in solution

of magnesium On to convert

kinase

(mM)

by

5 mM

50 mM imidazole buffer, 100 mM ADP, pH 7.5, 30’. V, = velocity in presence of 5 mM ATP.

Conditions:

part

of pyruvate magnesium.

this

is in

is present, basis, most

a 2 mM

of it into

the

form

while

ADP

concentration Mg-ADP,

ATP

KCI, 0.37 without

of its magnesium for

782

salt

a 10 to 20-fold of ADP

the

16

true

would

substrate

at various

mM ATP,

when

concentrations

PEP, 0.25 mM V5 = velocity

an equimolecular

excess require for pyruvate

of magnesium at least kinase.

is

20 mM

mag-

If ATP

Vol.

31,

No.

5, 1968

BIOCHEMICAL

AND

Table Inhibition

of Pyruvate

a)

glycylglycine

10 mM

Kinase

by ATP

buffer,

mM

RESEARCH

COMMUNICATIONS

2

Under

100

BIOPHYSICAL

the

Conditions

KCI,

10 mM

of Reynard

MgC12,

et al,

0.37

mM

(1961)

PEP,

at 30’.

2 mM

ADP

Rate {ODu/min

b)

pH 8.5 pH 8.5

no +

ATP 5mMATP

893 346

2.6

pH 7.5 pH 7.5

no +

ATP 5 mMATP

758 382

2.0

10 mM

glycylglycine no

ATP

pH

+

5 mM

ATP

ATP

/ rate

8.5

et al,

is added

and

complexes

conditions

Lowry

and

It seems

by a competition

would

listed

clear

had

in Table

3 when

similar

system

was

pH of the

ATP.

mixture

low

t Va/V5

7.0

as calculated

who

resl;lts the

first

for

series

the

from

the

however

not

the

authors

results

changed

2),

it is worth make by the

et al.

only

results

no mention addition

that

of ATP

and can

(1966),

be explained and

HP04

= 76)

which

in the

above

terms

is more

inhibition

in the

second was

could

the buffering

with

Cohn

Lowry

concentration

of precautions

tabulated

Mg

et al. ADP

to fall.

are

Furthermore,

exptanation

the

inhibition

(1961)

for

of the

be expected

Mildvan

et al.

constant

a small

noting

783

(1967),

of Reynard when

concentration

can

ATP

present.

An

was observed (Table

reported

(formation

the

the rate

of Reynard

magnesium

present

then

and

have

of Tanaka

the

and

of magnesium

be reduced

to explaining

experiments

was

will

magnesium.

conditions, very

amount

available

inhibition

In my own

under

ADP

phosphate

the

it comes

the

and

with

5 mM

reduce

when

mM.

5 mM

of workers

that

(1964),

of ATP

also

further

difficult

with

species

by a number

Passoneau

Passoneau

1.5

200

an equal

Mg-ADP

used

3.

10mMMgC12,0.12mMPEP,0.2mMADP 300

with

active

in Table

KCI,

1.3

(1961).

catalytically The

1OOmM

pH 8.5

Reynard

0.2

buffer,

= ra t e without

*vo/v5

./ml)

as low

be obtained

capacity to ensure

a resulting

series

fall

of their that

the

in rate.

as

of

2 A

Passoneau

I PEP and

(1964).

ADP

concentrations

at potassium

et al . (1967)

and

* Kf present

Tanaka

Lowry

ibid

not

clear

pH

7.5,

the

also

inhibition

B.5,0°

B-5,0”

account

present,

7.0,28’

published

pH 7.5,25” K2HP04

from

tris,

imidazole*,

glycylglycine,pH

ATP

29”

pH

which

temperature

under

glycylglycine,pH

tris,

and

5 mM

50 mM

20 mM

10 mM

10 mM

acetate,

(ii)

(i)

et al.

Reynard

(1961)

50 mM

(1966)

and

Mildvon

Cohn

Buffer

Authors

Conditions

100

150

100

100

100

CK+l mM

but

0.1

0.05-0.4

0.01-0.1

CPEPI mM

believed

was brain

2+

measured.

5

5

10

10

4

mM

l-10

3

o-7.5

o-7.5

1.25,2.5

I ATPI mM

experiments

cMg++1

as in the other

kinase.

2+

0.14

0.2

2

2

CADPI mM

has been

to be 2 mM

pyruvate

kinase

0.002-0.02

of pyruvate

3

enzyme

Table

described.

3.5

1 .o

0.14

0.12

0.33

mM

Ki for

ATP

Vol.

3 1, No.

5, 1968

BIOCHEMICAL

lt is significant PEP reported 2.7

by

x 10s4

different

M,

Km for

a 30-fold

range

(Table

inhibition

as suggested

with

the

brain

that

enzyme

although

workers

Reynard

of the

in a narrow

range

to 7.5

that

the

RESEARCH

Michaelis (Km

x 10s5),

specifically

that

for

ATP

for

ADP

and

ADP

= 1.26

x 10e4

to

values

for

Ki

(ATP)

cover

not

a true

observed

Lowry

COMMUNICATIONS

constants

their

inhibition

In fact,

et al.

quite

BIOPHYSICAL

values

x 10s5

3) suggesting

stated

the lie

PEP = 3.2

by

AND

was

and

Passoneau

was

not

competitive

in their

competitive

experiments

with

either

substrate. Summary The It was

inhibition

concluded

magnesium

of pyruvate

that

complexing

abolished

completely

the

kinase

inhibition

ATP was

reported

properties when

by

of ATP

sufficient

by and

previous

it was

magnesium

investigated

shown

was

workers that

under was the

various

conditions.

attributable

to the

inhibition

was

added.

Acknowledgements I am indebted Research

Council

to Mrs.

of Canada

Caryl for

Barron

financial

for

her

technical

assistance

and

to the Medical

support.

References 1.

Czok, p.

2. 3. 4. 5. 6. 7. 8. 9.

Lowry, McQuate, Melchior, Meyerhof, Mildvan, Reynard, 236, Ralleston, Tanaka, -62,

R. and Eckert, L., Methods of Enzymatic Analysis, Ed. by H.U. Bergmeyer 224, Academic Press (1965). 0 .H. and Passoneau, J.V., J. Biol. Chem., 239, 31 (1964). J.T. and Utter, M.F., J. Biol. Chem., 234, 2151 (1959). J.B., Biochemistry 4, 1518 (1965). 0. and Oesper, P., J. Biol. Chem., 179, 1371 (1949). A.S. and Cohn, M., J. Biol. Chem., 241, 1178 (1966). A.M., Hass, L.F., Jacobson, D. D., andBoyer, P. D., J. Biol. Chem., 2277 (1961). F.S. and Newsholme, E .A., Biochem. J., 104, 524 (1967). T., Harano, Y., Sue, F., and Morimura, H., J. of Biochem., (Tokyo), 71 (1967).

785