1973 Lienhard AP5A Inhibitor1121 3

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Gustav E. Lienhard and Isaac I. Secemski

Adenylate KinasePotent Multisubstrate Inhibitor of

-Di(adenosine-5')pentaphosphate, a5,P1PCOMMUNICATIONS:

1973, 248:1121-1123.J. Biol. Chem.

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Communication

THE JOURNAL OF BIOLOGICA~~ CHEMISTRY

Vol. 248, No. 3, Issue of February 10, pp. 1121-1123, 1973

Printed in U.S .A.

P,P-Di(adenosine-V)pentaphosphate, a

Potent Multisubstrate Inhibitor

of Adenylate Kinase

(Received for publication, October 20, 1972)

GUS TAV E. LIENHARD AND ISAAC I. SECEMSICI

From the Department of Biochemistry, Dartmouth Medical

School, Hanover, New Hampshire 03755

SUMMARY

Rabbit muscle adenylate kinase is potently inhibited by

PI, P5 - di(adenosine - 5)pentaphosphate (ApbA) but not by

the homologs of this compound with fewer phosphoryl groups

in the polyphosphate bridge and not by adenosine 5-penta-

phosphate. The inhibition by Ap& is competitive with

respect to both of the substrates, AMP and ATP. The

association constant for the binding of Ap& to adenylate

kinase is about 4

X

lo8 M-l tit 24 and pH 8.0.

Two substrate enzymatic reactions that proceed by way of a

ternary complex of enzyme and both substrates should be po-

tent ly inhibited by compounds that possess in an appropriate

spatial relationship within 1 molecule the binding determinants

for both substrates (1, 2). We have applied this principle to

identify a potent inhibitor of the enzyme, adenylate kinase (EC

2.7.4.3) from rabbit muscle. This enzyme catalyzes the trans-

fer of the terminal phosphoryl group from ATP to AMP. The

kinetic mechanism of this reaction is random bi bi (3), and it is

probable that the chemical mechanism invo lves direct displace-

ment of ADP from ATP by a phosphoryl oxygen atom of AMP

(4). The enzyme is probably a single polypeptide chain with

only one active site (5). This communication describes the po-

tent inhibition of adenylate kinase by the compound, P1,P5-di-

(adenosine-5)pentaphosphate

where A-O = adenosine with a bond to the 5 oxygen atom.

MATERIALS AND METHODS

Samples of calcium Ap,A,l calcium Ap3A, calcium Ap4A, and

sodium Ap& were most generously supplied by Dr. J. G. Moffatt

of the Institute of Molecular Biology at Syntex Research. Reiss

and Mof fatt (6) have previously described the synthesis and

characterization of these compounds. Ammonium psA was

kindly supplied by Dr. R. Lohrman of the Salk Institute for Bio-

logical Studies. Barium p4A was purchased from Sigma Chemi-

cal Co. and converted to the sodium salt by precipitation of the

* This research was supported by Grants GB 29205 and GB 33342 from

the National Science Foundation. Part of the research was carried out in

the Department of Biochemistry and Molecular Biology at Harvard

University.

1 The abbreviations used are: ApnA, P, PVli(adenosine-b)n-phos-

phate; p4A and psA, adenosine 5-tetra- and 5-pentaphosphate, respec-

tively.

barium from an acidic aqueous solution with sulfate . The iden-

tit y and purity of these compounds were checked by thin layer

chromatography upon polyethyleneimine cellulose (PEI-cellulose

F plates from EM Laboratories, Inc.) with aqueous LiCl o f

various concentrations as the solvent. The compounds showed

the expected mobilities relat ive to AMP, ADP, and ATP (7) and

gave only a single, ultraviolet light-absorbing spot, with the ex-

ception of psA, which contained about 20 p4A.

The purity of

the Ap,A was examined with especial care; the compound gave

a single spot (RF 0.55 to 0.68) upon chromatography in 2.0 M

LiCl of an amount that was large enough to allow detection of

5 of an ultraviolet light-absorbing impurity of different mobil-

ity. Dr. E. J. Griffiths of Monsanto Industrial Chemicals Co.

generously gave us samples of sodium tripolyphosphate and so-

dium tetra- and hexametaphosphates. Sodium polyphosphates

of average chain length 4 to 6 were purchased from Sigma Chemi-

cal Co. (P8385). Upon thin layer chromatography on PEI-

cellulose with aqueous LiCl as the solvent the linear polyphos-

phates migrated with the expected mobilities relative to each

other, and the same was true of the cyclic metapolyphosphates

(8). However, in contrast to the earlier report (8), tripoly-

phosphate moved more slowly than hexametaphosphate and

pyrophosphate moved more slowly than tetrametaphosphate.

Rabbit muscle adenylate kinase, pyruvate kinase, and lactic

dehydrogenase and yeast hexokinase were Boehringer Mannheim

products. Crystalline rabbit muscle fructose 6-phosphate kinase

was obtained from Sigma, and crystalline rabbit muscle creatine

kinase was a gift from Dr. L. Noda.

The initial velocities of the adenylate kinase reaction were de-

termined spectrophotometrically at 340 nm by coupling the

formation of ADP to the oxidation of NADH via the pyruvate

kinase and lactic dehydrogenase reactions (3). The reaction

mixture contained 50

m M

Tris-HCl buf fer, pH 8.0, 10 mM cys -

teine-HCl (freshly d issolved and adjusted to pH 8 with I.3 eq

of KOH), 0.5 mg per ml of bovine serum albumin (Sigma, crys-

tallized and lyophilized) , 1 m M potassium or sodium phosphoenol-

pyruvate, 10 mM MgS04, 0.1 m M NADH, ATP, AMP, inhibitor

(in some cases), 3 to 5 pg per ml of lactic dehydrogenase, 3 to 5

pg per ml of pyruvate kinase, and 0.022 to 0.035 pg per ml of

adenylate kinase. The reactions were initiated by the addition

of 5- or lo-p1 volumes of the inhibitor and the enzymes, in the

order given above, to the rest of the mixture after it had tempera-

ture-equilibrated at 24.0 in a l-ml cuvet te in the cell compart-

ment of the spectrophotometer. The concentrations of lactic

dehydrogenase and pyruvate kinase were sufhcient for effic ient

trapping of the ADP, since larger amounts of these enzymes did

not increase the rates. The concentration of adenylate kinase

in micrograms per ml given for the reaction mixtures is

based upon the absorbance of a stock solution at 279 nm (9) and

is equivalent to 1 .O to 1.7 X 10m9 M (10). The specific activ ity

of our enzyme was about 13 of that reported for the pure en-

zyme under similar conditions (11).

RESULTS AND DISCUSSION

The effects of various potential inhibitors upon the rate of the

adenylate kinase reaction are given in Table I . Ap5A is an

extremely potent inhibitor; it inhibits the reaction by 55 at

3 X 1w8 M. The rela tively weak inhibition (8 to 15 at 330 to

1121


3/4

1122

T ABLE I

Inhibition of adenylate kinase

For each inhibitor, the initial velocities of the adenylate kinase reac-

tion were measured in the presence and absence of the inhibitor, with the

same concentration of enzyme, by the method described in the text.

The concentrations of AMP and ATP throughout were 0.20 and 0.15

mu, respectively.

The per cent inhibition is 100 X (~0 - V~/ZIO, where

vi and 00 are the initial velocities in the presence and absence of in-

hibitor. resnectively.

APZA

AP

Apd

APEA

PSA

Pyrophosphate

Triphosphate

Tetrahexaphosphate

Tetrametaphosphate

Hexametaphosphate

a At 35.

Concentration

PM

10

10

1

10

100

90

0.03

0.10

1.0

10

12

12

10

10

10

10

10

-

_ _

-

* Assay mixture contained 88 my KCl.

~From R eference 12, which describes inhibition under condition s

almost identical with ours.

Per cent inhibition

o,b

oa,b

0*

d

47*-d

34c

55a.b

78J

98sb

1cnW

15

14

ii

0

0

0

d There is no inhibition by 200

calcium chloride.

I

IO

20

I/[AMP],mM-

FIG. 1. The kinetics of the adenylate kinase reaction in the pres-

ence (0) and absence (0) of ApA. The initial velocities were deter-

mined by the procedure described in the text. The velocities are expressed

as the decrease in absorbance at 340 nm per min (AA/min). The graph

to the

left

shows the dependence of the reciprocal of the initial velocity

upon the reciprocal of the concentration of AMP, with 0.15 rnM MgATP.

The concentration of Ap6A was 2.0 X lo* M. The graph to the right

shows the dependence of l/v upon the reciprocal of the concentration of

MgATP, with 0.20 mM: AMP, in the presence and absence of 1.0 X lo* M

Ap A.

The inset in each graph presents the values of l/r at the higher

concentrations of the variable substrate. The concentration of enzyme

in the experiments with varying concentrations of AMP w as about 1.6

times larger than that used in the experiments with varying concentra-

tions of ATP.

400 times higher concentrations) by pr,A and Ap4A demonstrates

that the requirements for potent inhibition are that there be two

adenosine groups and that they be linked by a polyphosphate

bridge with at least five phosphoryl groups. The spec ific ity of

Ap& for inhibition of adenylate kinase was shown by the fac t

that 10-S M Apa did not inhibit pyruvate kinase, hexokinase,

fructose 6phosphate kinase, or creatine kinase. These enzymes

were assayed under the same conditions and in the same way as

adenylate kinase, with each substrate at 0.2 mm, except in the

case of creatine kinase, for which the assay mixture contained

0.5 mM ATP and 5 InM creatine. The results of a kinetic study

of the inhibition of adenylate kinase by low concentrations of

Ap,A are presented in Fig. 1. The inhibition is clearly competi-

tive with respect to both AMP and MgATP2-; the plots curve

upwards at high concentrations of each substrate because there

is slight substrate inhibition (see Reference 3). The above find-

ings indicate that Ap& binds to adenylate kinase with one

adenosine group in the site that binds the adenosine group of ATP

and the other adenosine group in the site that binds the adenosine

group of AMP,

The association constant (KJ for the formation of the complex

(EZ) between adenylate kinase (E) and ApSA (I) can be esti-

mated in the following way. In the absence of inhibitor, with a

fixed concentration of MgATP2-, and at very low concentrations

of AMP, the total concentration of enzyme ([Elt) is approxi-

mately equal to the concentration of free enzyme ([Z&) and the

concentration of the complex with MgATPz- ([Es . .Mg-

ilTP30) ; also, the initial velocity of the reaction (~0) is propor-

tional to [E. + .MgATI-]o and to the concentration of AMP

(Equations 1 and 2; see Reference 3).

[El, = [E]o + ]E...MgATP2-]o

(1)

o0 = k [E.. .MgATPz-]o[AMP]

2)

Similarly, under the same conditions, in the presence of ApsA,

Equations 3 and 4 hold.

[El, = [Eli + [E.. .MgATP*]i + [EZ]

(3)

si = k [E.. .MgATPz-]i[AMP]

(4)

By use of these equations and the expression for the association

constant for the formation of E . e . MgATp (KMgATp), it can

be shown by straightforward algebra that

Ki

= 6 2 - 1

(K~~mp

lMgATP1 + 1)

( )

(5)

Vi

An identical type of expression describes the relationship between

Ki and the equilibrium constant (KAMP) for formation of the

complex between enzyme and AMP when the concentration of

MgATP is very low. Kuby et al. (9) have found that the values

of KLQ AT P and LMP

for rabbit muscle adenylate kinase are lo4

~-1 and 1.6 x 103 ~-1, respect ively, at pH 7.9 and 3. The value

of V&J; at very low concentrations of each variable substrate is

equivalent to the ratio of the slope of the plot in Fig. 1 for the

case with inhibitor present to that for the case with inhibitor

absent. Using the values o f KMgATP and KAMP, the values of

v, ,/ v; given b y the data in Fig. 1, and the two expressions for Ki,

we calculate values of 2.8 and 5.0

x

lo8

M-l

for KG

Although the binding of Ap,A to adenylate kinase is strong,

the ra,te of dissociation of the complex is relatively fas t. When a

solution of 1.0 x 10-e

M

Ap,A and 5 X lOA M adenylate kinase

(assuming all of the protein is the kinase) in 10 mM MgSOd-10 mM

cysteine-0.5 mg per ml of bovine serum albumin-50 mM Tris-HCl,

pH 8.0, was diluted by a factor of 500 into the usual assay mix-

ture that contained 1.0 mM AMP and 0.15 mM ATP, there was

no detectable lag in the establishment of the initial velocity ,

which was the same as that for the same dilution of a stock SO~U-

tion of the enzyme alone. The lack of a measurable lag requires

that the half-time for dissociation of the complex be less than

0.2 min.

The magnitude of the association constant for the binding of


4/4

Ap5A to adenylate kinase is about 20 times larger than the prod-

uct o f KMgATP and

KAMP.

Since the binding of each substrate

appears to strengthen somewhat the binding of the other (3),

Ki for ApsA is less than 20 times larger than the association con-

stant for the formation of the ternary complex from free enzyme

and both substrates. Wolfenden has termed such inhibitors

multisubstrate analogs (l), and this description of Ap,A seems

more appropriate than the term transition state analog for two

reasons. First, the transition state for phosphoryl transfer in

the adenylate kinase reaction probably resembles a trigonal bi-

pyramid with the phosphorus atom at its center and the entering

and leaving oxygen atoms at its apices (4) :

Clearly, the pyrophosphate functional group of Ap6A does not

closely resemble the pentaoxy phosphorus atom of the suspected

transition state.

Second, a good analog of this transition state

should have a much larger association constant for binding to the

enzyme than the association constant for the formation of the

ternary complex from enzyme and both substrates (1, 2).

Ap& may prove to be useful for studying the role of adenylate

kinase in metabolism (4) and also for inhibiting adenylate kinase

wherever its presence complicates the investigation of other reac-

tions of the adenine nucleotides, such as in the investigation of

1123

the role of the ADP-ATP exchange reaction in oxidative phos-

phorylation (13). It will probably be possible to prepare potent

inhibitors of other phosphoryl transferring enzymes (14) by re-

placing the phosphoryl group that is transferred by a pyrophos-

phate bridge.

Ackrwwledgmen&--We thank Dr. Lafayette Noda for sharing

his knowledge of adenylate kinase with us.

1.

2.

3.

4.

5.

;I

8.

9.

10.

11.

12.

13.

REFERENCES

WOLFENDEN,

R. (1972) Acets. Chem. Res. 5, 10

LIENHARD, G. E. (1972) Annu. Rep. Med. Chem. 7,249

RHOADS, D. G., AND LOWENSTEIN, J. M. (1968) J. Bill. Chem. 243,

3963

NODA, L. (1973)

in

The Enwmes, (BOYER, P. D., ed) Vol. 8, 3rd Ed,

Academic Press. New York, in press

WEED S, A. G., AND

NODA,

L. (1968) B hem J. 107,311

REISS, J. R., AND MOFFATT, J. G. (1965) J. Org. C m. 30.3381

RANDERATH,

K.,

JANEWAY,

C. M., STEPHENSON, M. L.,

AND ZAM-

ECNIK,

P. C. (1966) B hem. Btiphys. Res. Commun. 24,98

TANZER, J. M., KIUCHEVSK~, M. I., AND CH~SSY, B. (1968) J. Chro-

ma n-. 38,526

KUBY, S. A.,

MAEOWALD,

T. A.,

AND NOLTMANN, E.

A. (1962) Bio-

chemistry 1,748

NODA, L., AND KUBY, S. A. (1957) J. Bill. Chem. 226,551

NODA, L., AND KUBY, S. A. (1963) Methods Enzgmol. 6,223-230

PURICH, D. L., AND

FROMM, H. J. (1972) B him. Btiphys. Acta 276,

563

PULLMAN, M. E., AND SCHATZ, G. (1967) A nnu. Rev. B hem. 36,

539

14. MORRISON, J. F.,

AND

HEYDE, E. (1972) Annu. Rev. Bb-

them. 41.29

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1973 Lienhard AP5A Inhibitor1121 3