Eur. J. Biochem. 133, 169-172 (1983)
,(3FEBS (1983)
The ap- Methylene Analogues of ADP and ATP Act as Substrates for Creatine Kinase
AGOfor this Reaction and for the Hydrolysis of the aD-Methylene Analogue of ATP
E. James MILNER-WHITE and David S. RYCROFT
Departments of Biochemistry and Chemistry, University of Glasgow
(Received November 15, 19821February 28, 1983) - EJB 6198
The ap-methylene analogues of ATP and ADP, [ajCH2]ATP and [afiCH2]ADP, are substrates for creatine
kinase. However, the rate of the phosphoryl transfer reaction catalysed is about 10-5-times lower than that with
normal ATP. The affinities of the analogues (especially [crOCH2]ADP) for the enzyme are lower than those of the
normal substrates. The equilibrium constant at 25 -C, measured using 31PN M R , for the reaction Mg[ajCH2]ATP
Mg[ajCH2]ADP + phosphocreatine + H + is 2.2 x 10-l' M compared with a value of
+ creatine
2.5 x 10- l o M for the same reaction with the normal substrates, corresponding to a difference in AGO values of
11.7 kJ . mol-'. It follows that AGO for the hydrolysis of the terminal phosphate group of Mg[@CH2]ATP is
less favourable by 11.7 kJ . mol-' than that for MgATP.
The xP-methylene analogue of ATP ([ajCH2]ATP) is a
stable analogue of ATP which is identical except that it
possesses a methylene group (- CH2-) instead of an oxygen
atom between the c( and p phosphorus atoms. [ajCH2]ADP
is the corresponding analogue of ADP. [a/EH2]ADP was
first synthesised by Myers et al. [I] and both have been commercially available for some years. I n general (2, 31 it seems
that these analogues can often bind to ATP or ADP binding
sites of enzymes [4-11] yet only for a few enzymes have
reactions involving the transfer of the 7-phosphoryl group
frotn the ATP analogue been demonstrated. Chloroplast
coupling factor [12] can hydrolyse [afiCH2]ATP; the rate of
the reaction is lower than that for ATP. The ATP analogue
is also a substrate for the ATPase of myosin [13]. Although
the steady-state rate of hydrolysis is similar to that of ATP,
rapid reaction studies have shown that the initial rate of
cleavage is about 1000-times slower than with ATP [14,15].
It will be shown in this paper that creatine kinase catalyses
phosphoryl transfer between the ADP analogue and creatine
and that the equilibrium constant with the analogue is substantially different from that with the physiological substrates.
EXPERIMENTAL PROCEDURES
Materials
Creatine kinase was purified from rabbit back and leg
muscle as previously described [16,17]. The purity and specific
activity of the enzyme was as before [17]. Concentration of
the enzymes was carried out as previously [IS].
Abbreviations. [a/3CH2]ADP, the ap-methylene analogue of ADP,
i.e. adenosine 5'-[x,fi-methylene]diphosphate;[xfiCHz]ATP, the xf imethylene analogue of ATP, i.e. adenosine 5'-[a,/f-methyiene]triphosphate; [ByCHzIATP, the By-methylene analogue o f ATP, i.e. adenosine
5'-[/3,y-methylene]triphosphate;Bicine, N.N-bis(2-hydroxyethy1)glycine;
5es, 2-[bis(2-hydroxyethyl)amino]ethanesulphonic acid.
Enzymr. Creatine kinase (EC 2.7.3.2).
[c(PCH2]ATP, [xjCH2]ADP and [PyCH2]ATP were purchased from Miles Biochemicals (Stoke Poges, Slough, UK).
ATP, A D P and phosphocreatine were from Sigma Chemical
Co. (Fancy Rd, Poole, Dorset, UK). Some later samples of
the ATP and ADP analogues were from Sigma. All other
chemicals, including Bicine and Bes, were from BDH Chemicals (Poole, Dorset, UK).
Enzyme Assays
A pH-stat assembly was used as described previously [17,
191 to monitor both the forward and backward reactions of
creatine kinase and its ATPase activity. The forward reaction
and the ATPase activity were followed by the addition of
NaOH (5 mM) to maintain the pH. The backward reaction
was followed by the addition of perchloric acid (5 mM).
Calculution OfK, and Ki VulucJs
K,,, values were determined from Lineweaver-Burk plots.
The values given are those for one of the substrates at a fixed
concentration of the other, as noted in the legend to Table 2.
Ki values were also determined from Lineweaver-Burk plots,
taking advantage of the observation [20] that the value of
Km/V alters by a factor of (1 + i / K J for both competitive
and mixed inhibition.
31PN M R Spectra
Proton-noise-decoupled 3 ' P N M R spectra were obtained
at 40.5 MHz as described previously [18]. Chemical shifts are
expressed as positive to low field of external 850/0 (w/w)
HjP04.
Quantification of substrates from 3 1 P N M R Spectra
The relative concentrations of the various substrates were
determined by computing the areas under the peaks of the
170
N M R spectra. Proportionality between areas and conccntrations may not hold over a wide concentration range
or between different substrates. Therefore a solution was made
up (sample b in Fig. 1 and Tablc 2) containing known substrate
concentrations in approximately similar ratios to those being
measured in the equilibrium mixture (sample a in Fig. 1 and
Table 2) and scanned for 31Presonances under similar conditions. As the equilibrium mixture contained a very high
concentration of protein, a similar concentration of creatine
kinase that had been totally inactivated by limited protcolysis
[21, 221 was added to sample (b). In order to ensure compatibility of solutions the creatine kinase in sample (a) was also
inactivated before the NMR spectrum of the sample was run.
The ratio of the concentrations of the ED-methylene nucleotides
were calculated both from the ratios of the cx pcaks and from the
ratios of the fi peaks in the two solutions. The ratio of the
concentration of [aBCH,]ATP to that of phosphocreatine was
calculated from the ratios of the y peak of the nucleotide
analogue to the phosphocreatine peak in the two solutions. The
concentrations of inorganic phosphate, and also of the components of sample (c) in Fig. 1 and Table2, were calculated
assuming that their integrals were proportional to those of the
[cxP C H ,]AT P.
where x = bound substrate a s a proportion of the total
activc-site concentration, s = concentration of free substrate whose binding is being considered, i = concentration
of free substrate whose binding competes with the substrate
being considered.
Binding constants (both K, and Ki values) were taken as:
phosphocreatine. 2.0 m M : creatine, 9 mM ; Mg[abCH2]ADP.
4.2 m M ; Mg[xflCH2]ATP, 1.6 niM. These values are from
Table I . Each of the calculations was repeated using the
values for the free ligand concentrations that had been
calculated previously. However, this did not affect their final
values significantly.
RESULTS A N D DISCUSSION
Thc Eri,-j,nic-Catal~.scrlRtwctioti
iL?th
the Annlogue.~
The 31Presonances of each phosphorus atom of the z/J'mcthylene analogues and of ATP and ADP are well separated
from each other and from those of phosphocreatine (see Fig. 1).
The proton and magnesium ion binding effects on the 3 1 P
NMR spectra of the analogues are also known [23,24]. Their
pK, values lie within 0.5 of those of ATP and ADP.
On mixing together Mg[a/KH,]ADP and phosphocrcatine
(10 mM each) at 25°C at an initial pH of 9.0 in the presence of
creatine kinase (10 mglml), extra signals that correspond to
those of Mg[aPCH,]ATP gradually appeared. At very high
enzyme concentrations (300 mg/ml) after 1 h at 25 ,C only a
little of the ADP analogue remained. The pH values of these
solutions were also observed to fall slightly. In order to test
whether the reaction was enzyme-catalysed a similar mixture
Metlzod i f ' Correcting Eqiiilihriim Constunts to Allon.
f o r Biriding of Suhstrritos or Suhstrute Ann1npc.r
The equation used is :
Table 1. Sonic purnriirfcr.s of' reucrions cutalysed h j , c~reutinc~
kintrsr
The forward reaction was carried out at pH 9.0. 25°C in the presence of cy3teine (1 mM). Except where varied, the concentrations of thc substrates
were: creatine. 40 niM: ATP or the analoguc, 4 m M : magnesium acetate was added so that the free Mg2+ ion concentration was 1 mM. The
enzyme concentration with the analoguc was 21 mgjml. The backward reaction was carried out at p H 7.0, 25' C, in the presence of Bes (1 mM) and
cysteine (1 mM). Except where varied, the concentrations of the substrates were: phosphocreatine, 1 0 m M ; ADP or the analogue, 4 m M ; magnesium
acetate was added so that the free ME'+ was 1 m M . The enzyme concentration used with the analogue was 1 mglml. For the determination of K ,
values five different concentrations of the varied substrate were used. Assays in the presence and absence of the inhibitors were carried out using at
least three different values of the MgADP concentralion. The inhibitors, which werc all found to be approximately competitive, were added at a
concentration o l 8 niM. The equilibrium constant for the analogue was mcasured at 25 'C at pH 8.79 in Bicine (25 mM) buffer adjusted to the correct
pH with NaOH. Creatine and [afiCH,]ATP were mixed together with creatine kinase (20 mgiml). The final concentrations of the substrates are given in
Table 2. The equilibriuni constant quoted for the physiological substratcs [26] was measured at 2 5 ' C in glycylglycine,NaOH buffer (I = 0.1 M).
The maximum value for the [r[K:Hr]ATPase was calculated froiii the difference in the phosphate concentrations in the samples used in Fig. l a and b.
Thc value for thc rate of the ATPase activity at 25 C was measured with MgATP (4 m M ) and magnesium acetate (5 mM) at pH 9.0. in the presence of
cysteine ( 1 niM). n.d. = not determined
~-
Reaction
Parainetcr
Unit
Value with sub\trate
-
Forward reaction
Backward reaction
ATPase (or [a[CH,]ATPase)
Phosphorylation of creatine
spccific activity (initial rates)
K,,, for creatine
specific activit) (initial rates)
no added sodium salts
plus sodium acetate (0.1 M )
plus sodium nitrate (0.1 M )
without magnesium ions
K , for ADP or its analogue
K , for phosphocrcatine
K , lor [x/KHz]ADP
K , for [x/lCH2]ATP
Ki lor [&CHZ]ATP
specific activity (initial rates)
K
pmoI min-' (mg protein)
mM
pmoi i1iin-l (mg protein)-'
-
[cr/lCHz]ADP or
[x~~CHZIATP
ADP or ATP
1.7x!V3
84
n.d.
31 x lo-3
23 x l o - "
15r
in M
0
4.2
in M
2.0
mM
mM
in M
pmoi min-' (mg protein)-'
M
<4x 1 0 P
2 . 2 x 10-1'
9
40
42
29
0
0.14
2.1
8
1.6
I .2
1.3 x lo-'
2.5 x 10-'0
171
was incubated in the absence of enzyme. N o new signals
appeared. These observations suggested that creatine kinase
catalyses phosphoryl transfer between [a/XH2]ADP and
creatine.
The catalytic activity of the enzyme thereby inferred to
occur with the analogues as substrates was also detected by
using a pH-stat assay. Table 1 gives a comparison between
some parameters of the normal enzyme-catalysed reaction
and those with the analogues. Some other data are also included in the table. It should be noted that the values for the
forward reaction with the analogues were found to be of low
accuracy because of the high concentration of enzyme protein
that had to be used. Therefore it was not possible to dctermine
their K , values.
DcJterniination of Equilihriiim Constants f ; tlir
~ Reaction
with tlic Analogues
On mixing together Mg[apCHz]ATP, creatine and creatine
kinase, N M R signals due to Mg[ajZHz]ADP and phosphocreatine were detected (see Fig. 1). I n the absence of enzyme
no products were formed. In a series of preliminary experiments the amounts of the products formed, even with high
concentrations of enzyme, were lower than expected from the
equilibrium constant ( K ) for the physiological substrates.
This was found not to be due to enzyme inactivation. A possible explanation was a different K with the analogues as substrates. With some knowledge (see Table 1) of the rates of
the initial velocites it was possible to make up a mixturc that
was expected to come to equilibrium. In order to achieve
measurable amounts of Mg[aPCHz]ADP and phosphocreatine in this mixture, it was necessary to mix Mg[a/KH2]ATP and creatine together at high concentration over a long
period and at the highest pH that is compatible with the
activity of the enzyme. Full details are given in the legend
to Table 2 and in Experimental Procedure. The spectra are
in Fig. I . It should be noted that the commercial sample of
[a/KH2]ATP (see Table 2, sample c) contained small amounts
of [aPCH2]ADP for which corrections have been made.
An aliquot of sample (a) in Table 2 that had been incubated as the equilibrium mixture for 48 h was assayed by the
pH-stat assay and was found to have retained the full specific
activity of the purified enzyme with the physiological substrates.
Table 2 lists the calculated concentrations of the phosphorylated species present in thcse mixtures. It will be observed
that thc concentration of [ x P C H ~ J A D Pin sample (a) was
considerably greater that that of phosphocreatine. Two factors may account for this. One is that [aDCHz]ADP was
prcsent as an impurity in the [aPCH2]ATP sample (see
sample c in Fig. 1 ) . The other is that some secondary reactions,
presumably enzyme-catalysed, may have lead to the loss of
phosphocrcatine. For example, although ATP and ADP were
not detectable in the rP-methylene nucleotides used, trace
amounts could have been present. These would have exTable 2. C'onc~mtrationsof mokecu1r.c. in the so1ution.s used fbr tlze N M R
.spc)ctra of&. I
Sample (a) contained initially: [a/lCH2]ATP. 28 mM : magnesium
acetate, 32.5 m M ; creatine, 59.3 m M : creatine kinase, 20 mgiml; and
Bicinc, 25 mM, made to pH 9.0 with NaOH initially; in a volume of
1.2 ml. The mixture was incubated at 25 C for 48 h in a stoppered tube.
The pH after this period was 8.79. The reaction was terminated by the
addition of proteinase K (24 pg). Sample (h) contained the same mixture
t i 3 sample (a) except that no protein was added initially; it was incubated
at 25 'C for 48 h in a stoppered tube. To this was added creatine kinase
(24 iiig) that had been reacted for 30 min at 25 'C with proteinase K
(24 yg). Creatine phosphate (1.30 pmol) and [aPCH?]ADP (1.30 pniol)
were then added to give a final volume of 1 .2 mi. Sample (c) contained the
same mixture as sample (a) except that n o enzyme was added; it was
incubated for 48 h at 25 C . No enzyme protein was added to it suhscquently. All solutions were adjusted to pH 8.8 with either NaOH or
acetic acid before placing in the N M R spectrometer. Values given are
thosc of the total concentrations which include molecules that are bound
to protein; they include corrections for the presence of impurities in the
[N/CH~]ATP
~
M olec n le
Concentration
in
solution
~
+24
+19
+14
+9
+4
-1
-6
-
-11
(a) equilibrium (b) mixture of
mixture
cubstlate
analogues at
known
conLentration\
-16
&(P P m 1
V
Fig. 1. " P N M K .sliectro of so1urion.s t i ) niirc~hM R [ x / i C H , / A T P in tlw
prrsence und uhwnw (fc'reatine kinuse and vurious Iigunds wii s udrli,rl.
Samples were incubated in Bicine (25 mM, made initially pH 9.0 with
NaOH), magnesium ions added to a concentration 4.5 mM ahove that
of the nucleotides present and (a) an equilibrium mixture of [rrpCH~]ATP, crcatinc and creatine kinase with the reaction terminated by the
addition of proteinase K ; (b) a mixture of known concentrations of
[a/iCH2]ATP. [x/KH2]ADP, creatine. phosphocreatinc and creatine
kinase inactivated by proteinasc K ; (c) [a/KHz]ATP (as supplied) and
creatine alone. Full details are given in the legend to Table 2. The chemical
shifts of the phosphorus atoms under these conditions arc: Mg[z/KH*]ATP: x. 18.4 ppin; /I, 9.4 ppin; 7 . -4.8 ppin: Mg[xPCH2]ADP: x.
22.8 ppm; /?, 12.5 ppm; phosphocrcatine, -3.0 ppm; inorganic phosphate. 2.7 ppm. A few unidentified impurities are also evident
(c) mixture of
[r/lCH2]ATP
and creatine only
in M
[x/lClI2]ATP
[cr[KHz]ADP :
from a peak
fi-om /I peak
average
Phosphocrcatine
Inorganic
phosphate
"
26 8
2.29
2.12
2.20
1.01
0.92
indicatcs the amount added
28 0"
28 0
-
0.59
0.5s
0.57
none
-
1.08"
I .08"
0 71
0.54
172
changed with phosphocreatine and the ATP generated would
be expected to have been hydrolysed at a low rate. Creatine
kinase exhibits [25] an ATPase activity with a rate about
times that of the initial rate of the forward reaction with
physiological substrates. Another possibility is that there was
a low [sc[jCH2]ATPase activity. Table 2 shows that there was
some increase in the inorganic phosphate concentration which
could be explained by either of the reactions postulated above.
The equilibrium constant ( K ) for the creatine kinase
reaction with the nucleotide analogues calculated from the data
of Table2 is 2.25 x l o - ' * M. However, at the enzyme concentration used (0.49 mM active sites) a proportion of each of
the four substrates was expected to be present in the bound
form. This was estimated from the known K, and Ki values,
assuming that phosphocreatine competes with creatine and
that Mg[sc[KH,]ATP competes with Mg[abCH,]ADP. The
calculation is described in Experimental Procedures. These
values for the free substrate concentrations give a new value for
K of 2.24 x
M.
It can be concluded that K (25 " C ;I = 0.1 1 M ; free Mg2+,
M. This may be compared with a
4.5 mM) is 2 . 2 ~
M for the reaction with the physiological
value of 2.5 x
substrates [26]. The value of AGO for the creatine kinase
reaction in the direction of phosphocreatine formation is
thus 11.7 kJ . mol-' more positive with the Mga[l-methylene
analogues as substrates. A similar difference obviously applies
to the comparison between the values of AGOfor Mg[afiCHz]ATPase and for ATPase. Clearly the ap-methylene analogue
of ATP would not be such an effective 'high-energy compound'
as ATP, assuming that enzymes existed that allowed its phosphoryl transfer to be coupled to metabolism at a rapid rate.
The difference in the values of AGO may be connected with
free energy differences between the chelate ring of a niagnesiuni pyrophosphate and that of its methylene analogue, but
in thc absence of much knowledge of the structures involved
further discussion would not be meaningful.
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