Cyclocreatine phosphate as a substitute for creatine phosphate in vertebrate tissues. Energetic considerations

BIOCHEMICAL

Vol. 74, No. 1, 1977

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

CYCLOCREATINE PHOSPHATE AS A SUBSTI!I'UTE FOR CREATINE PHOSPHATE IN VERTEBRATE TISSUES.

ENERGETIC CONSIDERATIONS

Thomas M. Annesley Department

of Biochemistry,

Received

and James B. Walker

William

Marsh

Rice

University,

Houston,

TX 77001

November 2,1976 SUMMARY

The measured equilibrium constant ;f the reaction, P-creatine*+ cyclocreatine + creatine + P-cyclocreatine catalyzed by creatine kinase in the presence of a trace of MgATP, is 26 at $'O. P-Cyclocreatine has a V,,, 0.6% that of P-creatine and a Km of 3.7 mM, vs. 2.8 rnM for P-creatine, with chick breast muscle creatine kinase at pH 7.0. These results suggest that the high ratio of P-cyclocreatine/cyclocreatine (ea. 45) previously observed in breast muscle of cyclocreatine-fed chicks, compared to the much lower ratio of P-creatine/creatine (ca. 2) in ' the same tissue, primarily reflects free energy relationships between the synthetic and natural phosphagens. Rowley creatine is

et al.

(I)

have synthesized

and shown that

an excellent

Griffiths

(2)

to 60 mM P-cyclocreatine authors

suggested

cyclocreatine much lower

(ca. ratio

equilibrium

for

creatine

found

that

in the

(2)

that

45)

the

conditions

for

In this approach in the

breast

paper

we report

was to determine presence

Cyclocreatine kinase was purified

of creatine

(ca.

Reaction

1

e

MgADPl-

concentration

in support

the apparent

ratio

equilibrium

and a trace

1).

muscle.

compared

primarily

These

with

the

reflected

near-

2-

(1)

+ H+

+ P-cyclocreatine2kinase

in skeletal

of this

suggestion.

constant amount

up

of P-cyclocreatine/

+ P-cyclocreatine

of creatine

of

accumulated

of breast

muscle,

2),

+ creatine MgATP)

evidence

kinase

(Reaction chicks

water high

in chick

+ cyclocreatine high

in vitro

sarcoplasmic

(tr. of the

analogs

(cyclocreatine)

cyclocreatine-fed

unexpectedly

observed

+ MgATP*-

P-Creatine2in view

kinase

of P-creatinelcreatine

Cyclocreatine

of potential

l-carboxymethyl-2-iminoimidazolidine

substrate

and Walker

a number

(2) muscle. Our

of Reaction

2

of MgATP.

MATERIALS AND METHODS was synthesized as described elsewhere (2). Creatine from chicken breast muscle (3) to a S. A. of 250 Clmol/

Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.

185

Vol. 74, No. 1, 1977

BIOCHEMICAL

AND BIOPHYSICAL

I 0.5 ATP, MM

,

RESEARCH COMMUNICATIONS

I I .o

Fig. 1. Curve showing that attainment of the equilibrium of Reaction 2, catalyzed by creatine kinase, requires ATP. Initial conditions were identical to the first experiment of Table I. After incubation for 15 min, the creatine formed from P-creatine + cyclocreatine was determined colorimetritally. The equilibrium concentration would be 33.5 mM creatine.

min/mg protein at 300 and pH 9.0, assayed by the pH-stat method. Only one band was observed on SDS gels. Enzyme was stored at ho in 50 mM Tris.HCl, pH 8.0, and 14 mM mercaptoethanol. Calorimetric assays for the reactants of Reaction 2 were performed as described for the same four compounds in chick breast muscle (2). P-Cyclocreatine and P-creatine concentrations were obtained by substracting concentrations of cyclocreatine and creatine from the corresponding total values obtained after heating aliquots at 650 in 0.2 M perchloric acid for 15 min (P-creatine) and 45 min (P-cyclocreatine). Keq of Reaction 2 was measured after incubation of reactants for up to 8 hours at volume containing: Mg acetate, 2 mM; ATP, 1 mM; creatine 37O in 5 ml total kinase, 3 mg; and 50 mM of either triethanolamine.HCl, pH 7.0, or Na glycine, pH 9.0. Kinetic constants for creatine kinase were determined by coupled enzyme assays (a) in the forward direction by measuring ADP formed by NADH disappearance (4), and (b) in the reverse direction by measuring ATP formed by NADPH appearance (5). The reverse direction was also assayed by colorimetric determination of cyclocreatine or creatine formed (2). Initially cyclocreatine was chemically phosphorylated in this laboratory (by G. R. Griffiths) with Poll and NaOH and the product purified by column chromatography on Dowex-l(HCqactive isomer to be formed. 3 . We found only the enzymically for large scale synthesis (G.L. Kenyon, However, the use of LiOH is preferable personal communication). Cyclocreatine (45 g) was dissolved in 275 ml of 3 M LiOH and cooled to Oo with a dry ice-ethanol bath during the stepwise addition over 2 hours of a total of I20 ml of PoCl3 and 1000 ml of 3 M LiOH. The LiOH was added at intervals to keep the pH above 13. After 2.3 hours, the Li3PO4 and the supernatant solution was adjusted to pH was removed by filtration, 7.6 with 5 N HCl; 5 volumes of 95% ethanol were then added. The next day the fine precipitate was removed by filtration. The solution was cooled to -100 for 12 hours, then returned to room temperature to allow formation of crystals, which were filtered, washed with cold water and cold ethanol, and dried in a vacuum over P2O5. Yield: 25 g. The product was recsystallized from waterethanol, washed, dried, and stored at -100. The dilithium salt: (a) has an IR spectrum like that reported by Rowley et al. (1) for the enzymic product; (b) consists of only one active isomer as determined by coupled enzyme assay (5); (c) is hydrolyzed to cyclocreatine after 45 min at 650 in 0.2 M perchlo-

186

Vol. 74, No. 1, 1977

BIOCHEMICAL

TABIE I of Equilibrium Constant

Determination Conditions

AND BIOPHYSICAL RESEARCH COMMUNICATiONS

P-Creatine

Cyclocreatine

of Reaction

Creatine

2

P-Cyclocreatine

Keq

Initial Final

40 6

40 7

0 31

0 32

23.6

Initial Final

;

:

40 30

40 36

25.7

Initial Final

35 11

35 13

35 59

35 60

24.8

Initial Final

50 11

50 10

20 54

42”

20.6

Initial Final

5 5

5 5

25 27

25 27

29.2

bation

Concentrations (mM) of reactants for 6 to 8 hours at 370.

are

given

at 0 time

and after

incu-

ric acid (2); and (d) has pK,'s of approximately 3.1, 4.5, and 10.7. The active isomer is now known to be the 3-phosphono, rather than the 2-phosphono (l), derivative from X-ray crystallographic analysis (G. N. Phillips and F. A. Quiocho, in preparation)(cf. unpublished data cited in (6)).

Equilibration tine

kinase

provide tine

of the but

also

additional from

was selected

for

length

chick

MgADP),

against

breast

without

2 requires

as shown in Fig.

a double

muscle.

Keq determinations of time

RESULTS in Reaction

reactants

MgATP (or evidence

kinase

venient

four

displacement

An ATP (or in order

markedly

not

1. These

only results

mechanism

ADP) concentration

to achieve

perturbing

the

trea-

for

crea-

of 1 mM

equilibrium

in a con-

equilibrium

values

obtained. Table

I summarizes

five

separate

tions

of the

experiments four

was substituted We observed in the

the

physiological

data

at pH 7.0,

employing

reactants. for

that,

equilibrium

ATP, the like

The average average

P-creatine, range,

SO

Gq

for

value

Reaction different for

Gq was 28.4.

initial

from concentra-

Qq was 24.8.

When ADP

At pH 9.0 the

hq

none of the

pI&'s

of Reaction

2 should

187

2 obtained

was 25.7.

of P-cyclocreatine be pH-independent

occur

Vol. 74, No. 1, 1977

BIOCHEMICAL

AND BIOPHYSICAL

24

RESEARCH COMMUNICATIONS

-

02 I/

P-CREATINE

, u~-l

I/

0.4

0.6

P-CYCLOCREATINE

, MM-’

Fig. 2. Double-reciprocal kinetic plots for the reverse reaction catalyzed by chick breast muscle creatine kinase at pH 7.0 and 370. Incubation mixtures contained 37 mM Mg acetate, 11 mM ADP, and 29 mM triethanolamine.HCl, pH 7.0. P-Cyclocreatine: rC, = 3.7 mM; V, P-Creatine: & = 2.8 mM; V,,, =

over

a broad

range.

The overall

ATP or ADP and at pH 7.0 Kinetic muscle

data

creatine

3.7 mM for creatine

coupled

that

for

creatine

with

vided

chick

kinetic

parameters

catalyzed

reactants

by creatine

has provided

gives

the

kinase

(8)

1 obtained

corresponding

reaction

a value

of 26,

for

in agreement

188

2).

(cf.

from the

0.7$

by

of 55 m&f for

45%that

cyclofor

Substitution

relationships

when cyclocreatine on the

of 3.6 mM for

assayed

7).

of for

the

or creatine

I&q observed Haldane

with

creatine

with

the measured

of

ob-

P-cyclocreatine

a G

Haldane

Values

G's

cyclocreatine

kinase

check

Reaction

P-cyclo-

reaction,

indicated

respective

an independent

of Keq for

by Keq for

the

creatine

and a Vmax for

for

forward

and a V,,,

muscle into

and V,,

the

a &

and 37O were

7.0

breast

indicated

and 370 (Fig.

and 37O (4),

creatine

breast

for

by chick

method,

P-creatine

P-creatine, data

SD.

calorimetric

(5) at pH

of Keq, with

1 catalyzed

at pH 7.0

at pH 7.0

vs. 20 mM for

2. The ratio

P-creatine

kinetic

2 4.7

2.8 mM for

and 3.2 mM for

P-creatine.

substrates

by the

enzyme assay

enzyme assay

reaction

of Reaction

vs.

creatine

these

reverse

P-cyclocreatine

by coupled

for

was 25.8

assayed

P-cyclocreatine that

the

or 9.0,

kinase,

0.6%

tained

for

of 16 determinations

average

for

equation

and P-creatine Keq for

are

Reaction dias Reac-

BIOCHEMICAL

Vol. 74, No. 1, 1977

tion

2. In this

calculation

Mg,ATP and MgADP are

the

similar

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

assumptions

in both

were

made that

the

Km's for

reactions.

DISCUSSION This

work

thetic

was performed

phosphagen

difficult

from both

to determine

energy

of the

of P-cyclocreatine

relative

to ATP,

numerous

other

equilibria

Additional

would

(cf.

equilibrium fed range

10,ll).

(2),

is

thetic

lLq

for

relatively

natural

phosphagen

higher

than

resting

breast

by the

free

muscle

energy

relationships

For example, creatine larger

when

during total

reservoir

it

energy

pH over

the

rapid

growth,

to similar

cells

I)

less

physiological

nucleotides.

indicates

that free

of hydrolysis

(2)

The

this

energy

only

thus

2, without

of substitution

synthan

the

slightly in

can be explained

invoking

resting

of a major

by this prove

more stable

intracellu-

fast-twitch

during

of control 189

(2)

previously muscle

phosphate,

limited

portion

but a series

animals.

cells

the

interest. fed would

availability

of rapid

of the

synthetic

to be of considerable

in animals

that,

of high-energy

be kinetically

adenine

chicks

should

be predicted

an

of P-cyclocreatine/cyclocreatine

tissues

might

reservoir

would

compared

free

represents

muscular

equilibria.

consequences

of lower

2, which

reac-

of both

of Reaction

in vertebrate

of these

of cyclocreatine-

energy

ratio

ADP in pertinent

and during

chelated

of of

before

2 kcal/mol

a free

pK,'s

muscles

of cyclocreatine-fed

The physiological

phosphagen

i.e.

the

pH

breast

2 of 26 at 370 (Table

or additional

phosphagen

e.g.

involving

of ATP. The high

compartmentation

natural

independent

near

and unbound

Reaction

has approximately

pair,

that

tissues, hand,

at physiological

concentrations

in resting

constants

pair

Mg2'

is

free

such as determinations

equilibrium

other

attained

Reaction

phosphagen

if

in intact

On the

and dissociation

observed

lar

be involved

presumably

chicks

of free

It

1, and the

at pH's

complexities,

compartments,

to be estimated

involved

syn-

standpoints.

of Reaction

pH and concentrations

exercise

and kinetic

constant

intracellular

were

as a potential

equilibrium

MgATP and MgADP (8,g).

tants

P-cyclocreatine

the thermodynamic

the

of hydrolysis

because

to evaluate

cyclohave a of that

contractions,

Vol. 74, No. 1, 1977

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

ACKNCklLEDGMENTS This work was supported by grant C-153 from the Robert A. Welch Foundation, Houston, Texas. T. M. A. is a Predoctoral Fellow of the Robert A. Welch Foundation.

REFERENCES 1. Rowley, G. L., Greenleaf, A. L., and Kenyon, G. L. (1971) J. Am,. Chem. Sot. 93, 5542-5551. 2. Griffiths, G. R., and Walker, J. B. (1976) J. Biol. Chem. 251, 2049-2054. 3. Dawson, D. M., and Eppenberger, H. M. (1970) Methods Enzymol. 17A, 995-1002. 4. Bernt, E., Bergmeyer, H. U., and Mollering, H. (1974) in Methods of Enzymatic Analysis (H. U. Bergmeyer, ed.) Vol. 4, pp. 1772-1776, Academic Press, New York. 5. Lamprecht, W., Stein, P., Heinz, F., and Weisser, H. (1974) in Methods of Enzymatic Analysis (H. U. Bergmeyer, ed.) Vol. 4, pp. 1777-1781, Academic Press, New York. 6. Kenyon, G. L., Struve, G. E., Kollman, P. A., and Moder, T. I. (1976) J. Am. Chem. Sot. 98, 3695-3699. 7. McLaughlin, A. C., Cohn, M., and Kenyon, G. L. (1972) J. Biol. Chem. 247, 4382-4388. 8. Nihei, T., Noda, L., and Morales, M. F. (1961) J. Biol. Chem. 236, 32033209. E. A. (1962) in The Enzymes, 2nd Ed., Vol. 6, 9. Kuby, S. A., and Noltmann, pp. 515-603, Academic Press, New York. T. W. (1974) J. Biol. Chem. 249, 5845-5850. 10. McGilvery, R. W., and Murray, E. (1975) Biochem. J. 152, 173-180. 11. Sahlin, K., Harris, R. C., and Hultman,

190