Observation of Mg2+.ATP and uncomplexed ATP in slow exchange by 31P-NMR at high magnetic fields

Vol.

134,

No. 3, 1986

February

13,

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

OBSERVATION

IN

SLOW

OF

M.

Mg2+

Sontheimer:

*Max-Planck-Institut

W.

+Universitit

Bremen,

20,

UNCOMPLEXED HIGH

Kuhn,+&

ATP

MAGNETIC

H.

R.

FIELDS

Kalbitzer*

Forschung,

Abt.

Heidelberg, Fachbereich Chemie Bremen 33, FRG

D-2800 December

AND AT

medizinische 29, D-6900

fur Jahnstr.

Physik,

.ATP

BY 3'P-NHR

EXCHANGE

G.

Received

1379-1386

Pages

1986

Molekulare

FRG NW2,

1985 2,

SUMMARY: The 31 P-NMR lines of the B-phosphate groups in Mg .ATP and in metalfree ATP can be observed separately up to 280 K at 8.5 T and up to 285 K at 11.7 T. At 274 K and 8.5T the B-phosporous resonances are in slow exchange at pH values above pH 5, the y-phosporous resonances are in slow exchange only near pH 6, but in fast exchange at low and high pH-values. The fast exchange condition ho)$is for the a-phosphorous resonances over the entire pH-range. For Ca .ATP and metalfree ATP always fast exchange prevails dawn to the freezing pg);t of water even at 11.7 T. Based on the separate observation of the NMR signals of Mg". ATP and uncomplexed ATP new experiments are proposed and possible sources of error in ‘in vivo’ NMR studies are discussed. 0 1986 Academic Press, Inc.

Adenosine

triphosphate

metabolic

the

estimation

special ATP

is

is

ATP,

respective

the

the

Mg”.

phosphate

resonance,

interaction

where

true

ATP

factors

as

pH,

various

There

ligands

from should

very

may

a

valuable

in

vivo.

tool

have

sum

the

been

in in

ionic

shift

complex

strength,

influence

the

not

under

attempts

principle a

.ATP,

all

of

some

chemical

work

2+

the

area

the

the

Of

Mg

the

of

of

reactions

reliable

temperature,

most

concentration

represents

which not

the

Unfortunately,

concentration

is

in

as

biochemical

solution.

but

with

of

usually

system,

rule

concentrations

many

a method

essential

established

substrate.

in

determine

system

in

lines

complexes

been

nucleotide

3'P-NMR ATP

has

an

determination

the

different

well-defined

the

because

complex,

metalfree

P-NMR

of

interest

Mg*+.

the

31

pathways.

for

plays

(ATPI

b

and

chemical

to

of

the

in

a

g-

ological the hift

0006-291X/86

1379

AN

Copyright 0 1986 rights of reproduction

$1.50

by Academic Press, Inc. m any ,form reserved.

Vol.

134,

No. 3, 1986

position

in

often

BIOCHEMICAL

an

unpredictable

encountered in

center be

of

the

answered In

it NMR

to

enzymatic

show

will

Up

if

2+

Mg

to

now

complexes

this

COMMUNICATIONS

problem existing

recognized

by

the

question

active

could

only

methods. demonstrate

measure

ATP

substrate

study.

RESEARCH

biochemical

possible

the

kinetic we

BIOPHYSICAL

A different the

is

under

paper,

to

of

solution

indirect

possible and

which

enzyme by

this

is

way.

is

simultaneously

AND

that

directly .ATP

the

is

Mg

indeed

under 2t

the

.ATP true

suitable

conditions

concentration

by

substrate

in

3'p-

a given

reaction.

MATERIALS

AND

METHODS

and CaCl were analytical grade reagents. Adenosine-5'W12 triphosphate IA t PI and tris-(hydroxymethyl)-aminomethane ITrisl were obtained from Pharma Waldhof (Dusseldorf) and Roth (Karlsruhe), respectively. ATP was passed over a Chelex 100 column in order to remove divalent ions. The samples contained 5mM MgCl or 5mM CaC12, 1OmM ATP in 50 mM Tris-HCl, pH 6.4. For providing a 1 ock signal 020 ed to a concentration of 101. ";;,"4? P-NMR spectra were recorded in a 10 mm sample tube from Wilmad. (New Jersey) with a 6ruker HX-360 spectrometer operating at 145.6 MHz and a Bruker AM-500 spectrometer working at 202.5 MHz. The sample temperature was stabilized by a stream of dry nitrogen gas with appropriate temperature. All spectra are referenced to 85% phosporic acid as external standard. Spectra were fitted by a computer program based on the equations given by Gutowsky et al. (4) for a two site exchange of uncoupled nuclei, intermediate

previously

RESULTS

AND

ATP

resonance ATP

2

in

a

show

the

chemical

that

obtained

in

Mg2 +

concentrations

results

the

resonances

in

to

up

31

P-NHR

of

of

description (1).

exchange e.g. are

in

the

spectrum

in

K

(Fig.

characteristic

1380

2,

2.5 by

21.

ppm

At a

11.7

higher

metalfree The

agrees

well

with

to

infinite

8-

temperature. Mg 2i

Replacing of

in 11.

T the

2*

T the

(Fig.

extrapolating

2).

to

K

Mg

6.5

and

.AlP

260

1 and up

At

Hg

of

separated

containing

temperature.

domain ref.

285

solution

approximately

difference

fast

a

groups to

shift

approximately a

spectra

g-phosphate

(see

phosphorous up

an adequate coupled spins

for

a function

separated

correspondend

namely

represent even

P-NMR

as

1:2 of

clearly

31

the

ratio

lines

are

to domain

DISCUSSION

1 and

Fig.

and

shown exchange

fast

exchange

by

Ca

2+

even

at

Vol.

134,

No. 3, 1986

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

280 K e

274K

I -10.0

Fig.

" P-NHR temperatures.

1

spectra

at

‘,

Sample composition: artificial line

magnetic point

freezing the

much

The

effect

of

uncomplexed

lines

are

magnitude

of

metalfree

and

8-,

largest

a variation

of

and

the

6.7,

is

shown

and

tlg

10 mtl broadening

ATP,

5mt4 HgC12. used.

T and

at

shown).

reported

for

pH

on

in

fast

Fig. the

prevails

groups. Aw are

For

to

be

of

Mg

over

the

.ATP

1381

the of

a-

of

the

For

6.

entire

and

yabout

metalfree

Mg '+.ATP

g-phosphorous

the

lines

differs

a-

6, ATP.

The

in

for

resonances pH

y-

pH-range.

resonance

pH and

at and

the

pH

the

l1,3).

of

lines around

expected

2,

5

with

complexes

spectra

8.4.

the

consistent

.ATP

pH

pH

near is

Ca " P-NMR

function

different

50 mH Tris-HCl.

This

Aw of

a

at

temperature

Above

separation 1s

.ATP

resonance

exchange

ATP

pK--values

3.

distinghished

frequency

31

the

*

2t

low

a

not

be

only

y-phosphate

the

ATP

whereas

complexed

separations and

rates

lines

T of

(spectrum

exchange

can

I * * -20.0

11.7

separated,

groups

phosphorous

of

water

ATP

well

phosphate

5.3

of

higher

and

a-.

field

”

8.5

No

a hrgh

”

PPH

the

the the

mean

respectively

of

Vol.

134, No. 3, 1986

Fig.

2

BIOCHEMICAL

31 P-NMR temperatures.

at

spectra

11.7

Sample composition: No artificial line

(21. to

For be

the

the

g-

expected

at

modulation as

temperature model

calculated

kJ/mol reported

be (see from

(9.9

k-,

Vasavada

TC

the

slope The et

large

does

al.

in results

.ATP

the

11).

1382

K

293

K

290

K

280

K

274

K

different

pH

separation

of

Mg 2+.ATP

too

much,

complex spectral

8.5.

is

complex.

If

this slow

to

plot

general

as

a

fast

lines

function

(Fig. agreement

of

with

activation

The

in

295

3).

Arrhenius

are

COMMUNICATIONS

mM Tris-HCl.

from

(Fig.

ATP

at

50

transition

Methods). the

pK

vary

not

Hg".

and

tt

RESEARCH

frequency

the

fitting

by

Hg

5mM HgC12, used.

the

Materials the

and

experiments

of

obtained

kcal/moll. by

our

in

ATP

than

determine

rate can

suitable

time

observed

BIOPHYSICAL

relatively higher

Aw should

of

dissociation

The

be

pH-values

T of

1OmM ATP, broadening

a

correlation

exchange

exchange

resonances

AND

a

energy 4)

the

as

with

can 41 those

Vol.

134,

No. 3, 1986

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

9.6

6.5

6.1

I

”

”

-10.0

Fig.

3

3’P-NtlR

spectra

Sample

at

6.5

T of

composition:

Temperature

215

1,.

I,.

ATP

1OmN ATP, No artificial

K.

I.,

-20.0

Pi% and

t4gZ+

5mtl

HgCl line

.ATP

at

, 50

pH-values.

different

Tris-HCl. used.

mti

2roadening

k is-'

I T

2000.. 1500.'

600 700 600

I

I

.

I

.

3.2

3.3

3.1

3.5

,

,

3.6

3.7

,

>

3.6

-3 10

Fig.

4

Arf?enius .ATP Mg

The of

plot complex

dissociation the

spectra

of

the dissociation as a function

rates measured

k

of

were ;i

6.5

1383

rate k-, the inverse

obtained T

(x1

and

.

f IIt-‘]

of

the

of

the

from

a line

11.7

T

(01.

temperature fit

T

Vol.

134,

No. 3. 1986

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

T

Kl

320 310 300 290 280 270 260

250

200

100

300

coo

600

500

700

>

,

600

900

1000 v,

IMHzl Fig.

5

Critical

temperatures

T as

function

of

the

field.

magnetic

The critical temperatures T are calculated as described in the text as function of the magnetic field go (in units of the respective proton resonance frequency). The lifetimes used were calculated from (A) the results of this study (solid lines) and from I61 the results published by Vasavada et al. ill (broken lines). Sample compasitron: IA) see Fig. 1, (8) 5mM t4gC12. 1Omtl ATP in 25mll potassium-Hepes buffer, pH 6.0.

It are

is

important

to

be

the

field

temperature

at

the

(lbw

~~1

which

(1)

what

a given

the

Since can

obtained

vary for

temperatures

magnetic

fast

of

two

to

slow

the

slow field.

rate

exchange

considerably, a different

llbw

to

our

TC/

be well

dependent

in

we namely

occurs

included

buffer

reason

that

expected is

we

effects

temperatures,

exchange

are

exchange

For

crrtrcal

g-resonances

the

= 5). and

at

dependence

temperature

solution al.

know

at

expected

computed

and

to

on

the

the

= 1) separated

buffer

the data,

Vasavada

calculations

et

(Fig.

5).

CONCLUSIONS 1.

results

Our

concentration areas fields

demonstrate of

of

ATP

correspondent

the and

Mg2'.

low

temperatures.

that and

it

is

possible

uncomplexed

ATP

g-phosphate

This

resonances

may

be

1384

important

to

measure

directly

the from

at

high in

enzyme

the

peak

magnetic

kinetics

Vol.

134,

No. 3, 1986

where

concentrations

the important.

the

association in

fast

concentration to

the

remaining

permits

only

Mg2*

is

often

is

complexes of

the

las

ligands direct

of

ATP the

or

of

adenylate

kinase

complexed

with bound

transferred

are

phosphorylated

relatively component

NOE

,but

to

linewidth high

may

the

for

IAw

misinterpreted

of

that

the

if

saturation

from in

well

separated

to

show

case

for

via either

the

ADP

from

in

the

nucleotide

the to

elongation in

g-

the

factor

TuI

enzymes

which

reaction.

(at

the

8.5

K as

seen

a baseline 1385

lines

two

p-phosphate

T Ibw

~~/=l

at

from

Fig.

4).

artifact,

a

bound

to

phosphates

1:1

as

preference

nucleotide of

(at

in

from

ATP

these more

of

enzyme

transfer

the

estimation

and

the

transfer

metalfree

unequal

305

or

metal

question,

resonances

uncomplexed

enzymatic

~~/
the

the

its

of

methods

from

linked

of

p-phosphate

saturation

the

of the

the

products

from

one

this

possible

not

the

temperature be

be

covalently

ratio

ATP:ATP

of

concentration

two-dimensional

free

or

answer

are

is

to

(e.g.

the

can

NOE

not

during

a Mg'*.

in

but

reaction, Mg2+

of

NMRI

transfered

would ATP,

area

concentration

Although

Because

or

one-

nucleotide

the

differ

Mg2*.

by

it

ATP

Examples

function

g.,

ATP

uncomplexed

phosphate

At

or

is ATP

shift

ATP

enzyme.

resonances

by

transfer

a

an

proposed:

Mg2*.

substrates

phosphorous.

4.

e.

and

the

.ATP

uncomplexed for

as

y-phosphate

2*

it

uncomplexed

chemical

Ca

Mg 2+.ATP

present.

substrate

be

the

relative

the

whether

pH)

ATP

enzyme

are

can

saturation Mg2+.

of

. ATP.

exchange

addition

In

determined,

uncomplexed one

from

in

Ca 2+.ATP

Simultaneously

fast

activity

the

2t

the

of

complexes

Ca

measure

concentration

experiments

suitable

to

from

Since

absolute

true

enzymatic

interacting.

possible

complexes

indirectly

calculated

the

question

the

is

COMMUNICATIONS

metal

of

ions

a

be

their

presence

determination Ca2+

and

molecules

it

RESEARCH

the

ATP

and

only

all

g-resoncance.

line

It

in

determine

corresponding

2.

of

BIOPHYSICAL

nucleotides can

exchange,

even

possible

they

constants

always

and

where

AND

free

of

but

are

is

BIOCHEMICAL

a

from

the The

broader the

or

Vol.

134,

No.

3, 1986

integration be

of

BIOCHEMICAL

the

remaining

AND

component

BIOPHYSICAL

a too low

RESEARCH

COMMUNICATIONS

ATP concentration

determined.

ACKNOWLEDGEMENTS We

thank

Prof.

Riiterjans

to use the AM-500

and

Dr.

Hanssum

for

the

opportunity

spectrometer.

REFERENCES 1 Vasavada,

J. Inorg. 2 Jaffe, E. 3 Eigen, M. I

Gutowsky, 1228-1234.

V., Ray, 6. D.. and Nageswara Rao, Biochem. 21, 323-335. K. and Cohn, M. (1976) Biochemistry and Wilkens, R. G. (1965) Adv. Chem. H. S. and Holm. C. H. 11956) J. Chem. K.

1386

9.

D.

(1960

17, 652-657. Ser. 49. 55-79. Phys.

25,

may