1973 Lienhard AP5A Inhibitor1121 3
-
Upload
hishamasemmazal -
Category
Documents
-
view
223 -
download
0
Transcript of 1973 Lienhard AP5A Inhibitor1121 3
-
8/10/2019 1973 Lienhard AP5A Inhibitor1121 3
1/4
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.
http://www.jbc.org/content/248/3/1121Access the most updated version of this article at
.JBC Affinity SitesFind articles, minireviews, Reflections and Classics on similar topics on the
Alerts:
When a correction for this article is postedWhen this article is cited
to choose from all of JBC's e-mail alertsClick here
http://www.jbc.org/content/248/3/1121.full.html#ref-list-1This article cites 0 references, 0 of which can be accessed free at
http://www.jbc.org/content/248/3/1121http://affinity.jbc.org/http://affinity.jbc.org/http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;248/3/1121&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/248/3/1121http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=248/3/1121&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/248/3/1121http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=248/3/1121&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/248/3/1121http://www.jbc.org/cgi/alerts/etochttp://www.jbc.org/content/248/3/1121.full.html#ref-list-1http://www.jbc.org/content/248/3/1121.full.html#ref-list-1http://www.jbc.org/content/248/3/1121.full.html#ref-list-1http://www.jbc.org/cgi/alerts/etochttp://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=248/3/1121&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/248/3/1121http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;248/3/1121&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/248/3/1121http://affinity.jbc.org/http://www.jbc.org/content/248/3/1121http://affinity.jbc.org/ -
8/10/2019 1973 Lienhard AP5A Inhibitor1121 3
2/4
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
-
8/10/2019 1973 Lienhard AP5A Inhibitor1121 3
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
-
8/10/2019 1973 Lienhard AP5A Inhibitor1121 3
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