AMERICAN JOURNAL BIOTECHNOLOGY AND MOLECULAR SCIENCES
ISSN Print: 2159-3698, ISSN Online: 2159-3701, doi:10.5251/ajbms.2011.1.1.30.38
© 2011, ScienceHuβ, http://www.scihub.org/AJBMS
A specific role of 43Ca in the enzymatic ATP synthesis.
Anatoly L. Buchachenko 1, Natalia N. Breslavskaya 2, Dmitry A. Kuznetsov 1,3 *,
Stanislav E. Arkhangelsky 3, Vladimir P. Chekhonin 3
1
N.N.Semenov Institute for Chemical Physics, Russian Academy of Sciences, 4 Kosygin St.,
Moscow 119991, Russian Federation
2
N.S.Kurnakov Institute for General and Inorganic Chemistry, Russian Academy of Sciences,
31 Lenin Avenue, Moscow 119991, Russian Federation
3
Department of Medicinal Nanobiotechnologies, N.I.Pirogov Russian State Medical
University, 1 Ostrovityanov St., Moscow 117997, Russian Federation
ABSTRACT
43
40
This is the first report ever on the nuclear spin selective Ca Ca isotope effect expressed in the
Calcium affected enzymatic ATP synthesis. Once the creatine kinase (CK) catalytic sites were
43
2+
loaded with Ca , the resulting enzyme activity goes up (2.0±0.3 – fold) compared to the CK40
directed ATP synthesis rate occurred in the presence of nonmagnetic, spinless, Ca nuclei. The
effect manifests itself predominantly within a high concentration range (Calcium chloride,40 mM)
indicating that the ATP synthesis is a spin-selective ion-radical process. Primary reaction of this
2+
3path is an electron transfer between the reaction partners, С a(H2O)n ( n ≤ 4) and Ca(ADP) ,
generating an ion-radical pair where the singlet -triplet spin conversion takes place as a result of
the nuclear spin selectivity and a consequent magnetic isotope effect. This, in turn, makes an
essential impact on the final reaction’s ATP yield. To the contrast, there is no any isotope effect
2+
found within a low Ca concentration range showing that the ATP synthesis proceeds in a most
common nucleophylic way. The submolecular mechanisms of phenomena described and their
possible applied significance are under discussion.
Keywords ATP synthesis, magnetic isotope effect, Calcium-43, creatine kinase
INTRODUCTION
A novel isotope effect that makes a truly dramatic
impact on enzymatic, magnesium dependent, ATP
synthesis was recently discovered: the specific
2+
activity level of enzyme possessing Mg enriched
25
with the Mg nuclei was found to be almost twicetriple higher than that of the very same enzymes
(creatine
kinase,
pyruvate
kinase,
alphaphosphoglycerate kinase, etc) but containing
24
26
spinless, nonmagnetic, nuclei
Mg and
Mg
(Buchachenko et al. 2005 a,b,c; Buchachenko, 2009)
These data are no doubt in a favor to a statement
that the ATP synthesis is a spin-dependent ionradical process. There is no difference in activities of
24
26
enzymes with Mg and Mg; this is an evidence for
the fact that the magnetic isotope effect operates
indeed in ATP synthesis instead of a classical, massdependent (kinetic) isotope effect .
Magnesium isotope effect in ATP synthesis was
shown to be a function of concentration of Mg2+ ions.
At low concentrations, there is no isotope effect
found. Once a concentration of Mg2+ ions exceeds
physiological (intracellular) level by 20−60 times, a
huge isotope effect appears. It means that the
additional ion-radical mechanism of ATP synthesis is
switched on to provide some extra source for the
ATP yield increase. (Buchachenko et al., 2008) The
starting reaction of this mechanism relates to an
electron transfer path from the complexes
Mg2+(ADP)2- or Mg2+(ADP)3- to the Mg(H 2 O) n 2+ ion
(Buchachenko et al., 2010). It generates the ionradical pairs where populations of both singlet and
triplet states as well as the rates of a singlet-triplet
spin conversion are controlled by hyperfine coupling
of unpaired electrons with magnetic 25Mg and 31P
nuclei due to a Zeeman interaction. Consequently,
the resulting yield of ATP is a function of the nuclear
magnetic moment and magnetic field. All these
effects were observed and specified in experiment
(Buchachenko et al., 2008 a,b) . Both nucleophylic
and ion-radical mechanisms coexists independently,
they are about to contribute additively into the
common
ATP
synthesis
multi-compartment
biochemical system. The former dominates in vivo,
the latter may be turned on artificially by the targeted
delivering of Mg (or, even better, 25Mg) to the heart
Am. J. Biotechnol. Mol. Sci., 2011, 1(1): 30-38
muscle in order to hyper-stimulate
suppressed ATP synthesis rate and hence
some pathologies related to the ATP
(Amirshahi et al., 2008 a,b; Rezayat et al.,
phosphocreatine/160 M ADP for 40 min. This time is
found to be enough for the yield of ATP (or creatine)
to reach a limiting value. In control tests, i.e. in the
absence of CaCl 2 , the yield of ATP was negligible as
well as in case of the ice-cold incubation
measurements.
the preto prevent
deficiency
2009) .
This present work deals with an effort to reveal
whether or not the enzymatic ATP synthesis is to be
also specifically affected by magnetic calcium ions.
The contribution of Ca2+ ions to enzymatic ATP
synthesis processed in intact eucaryotic cells seems
to be rather neglected due to some known
circumstances. Thus, Ca2+ ions are bound with the
kinases catalytic site compounds more weakly than
Mg2+ ions; besides, the calcium concentration in
mitochondria is by order of magnitude lower than that
of magnesium ions.
Two series of experiments were carried out. First, the
enzymatic activities of 40Ca-CK and 43Ca-CK were
measured with unlabeled phosphocreatine by LC-MS
detection of creatine as the CK reaction product
which is formed simultaneously with ATP in the same
sample incubated. Secondly, [32P]phosphocreatine
was used. The former approach includes a HPLCderived mass-spec analysis of the acetone-soluble
pool
of
the
post-incubated
reaction
mixture (Buchachenko et al., 2008) while the latter is
based on determination of the HPLC-monitored ATP
yield by measuring radioactivity of the newly
synthesized gamma-[32P]ATP.(Buchachenko et al.,
2005)
Natural calcium pool contains mostly nonmagnetic
isotopes with spinless nuclei 40Ca (96.6%), 42Ca
(0.65%) and 44Ca (2.1%); for brevity we will further
specify natural calcium as 40Ca. We have substituted
magnesium ions in catalytic sites of creatine kinase
(CK) with different, magnetic and nonmagnetic,
calcium ions. For this purpose, the only magnetic
isotope of calcium with an atomic mass equal to 43
(natural abundance of 43Ca is 0.135%, nuclear spin
7/2, magnetic moment 1.3 µ B ) was also employed.
These two samples of calcium creatine kinase
(further we will denote them as 40Ca-CK and 43CaCK, respectively) were shown to exhibit enormous
isotope effect in enzymatic ATP synthesis similar to
that found earlier for magnesium creatine kinases
24
Mg-CK and 25Mg-CK.( Buchachenko et al., 2008)
Theoretical calculations of the energy characteristics
with full optimization of geometry was performed with
the Gaussian-03 program package (Frisch, 2003)
using density functional theory (DFT) with B3LYP
three-parameter exchange-correlation functional and
conventional 6-31G* split-valence basis set.
(Foresman, 1996) For this purpose, the HP9077A
analytical unit has been employed.
RESULTS
Structure of Calcium Complexes: The idea of
search for the Calcium-43 related isotope effect in the
reactions of enzymatic ATP synthesis was in fact
stemmed from DFT calculations of the energy of
electron transfer reactions:
METHODS
40
CaCl 2 and 43CaCl 2 were prepared by treatment of
CaO (0.135% of 43Ca) and 43CaO (86.7% of 43Ca),
both purchased from the RAS Isotope Center,
Obninsk, Russia, with analytically pure HCl. The
impurities of metals (Na, K, Zn, Mg, Fe, Ni, Ba)
determined by atomic absorption spectroscopy did
not exceed 30-50 ppm in both 40CaCl 2 and 43CaCl 2
samples. Creatine kinase CK (E.C.2.7.3.2), 40.0 kDa
active monomer, purified from V. xanthia raddei
venom (Buchachenko et al., 2008; Kouznetsov et al.,
and [32P]phosphocreatine with 6,4002003)
7,200 Ci/mmol initial specific activity (from Amersham
Radiochemical Centre, UK) were also employed.
40
Ca(H 2 O) n 2+ + Ca(H 2 O) m 2+(HOPO 2 OPO 3 СН 3 )2- →
(1)
Ca(H 2 O) n + + Ca(H 2 O) m 2+(HOPO 2 OPO 3 СH 3 )Ca(H 2 O) n 2+ + Ca(H 2 O) m 2+(OPO 2 OPO 3 СН 3 )3- →
(2)
Ca(H 2 O) n + + Ca(H 2 O) m 2+(OPO 2 OPO 3 СH 3 )2The CK catalytic site – engaging
phosphorylation mode might be presented as:
For CK activity measurements, enzyme samples
were incubated at 37C in Mg-free media containing
Tris-HCl (pH 6.35)/CaCl 2 (either 43Ca or 40Ca)
10−160 mM/12.5 M ATP/80 g CK/15 mM
potassium
phosphate/160 M
ADP
Ca(H 2 O) n 2+
Ca2+(ADP)-
+
Ca2+(ADP)2(3)
→
Ca(H 2 O) n +
+
Ca(H 2 O) n 2+
Ca2+(ADP)2-
+
Ca2+(ADP)3(4)
→
Ca(H 2 O) n +
+
We have calculated the structure / energy patterns of
complexes 1 (Chart 1) in which Ca2+(ADP)2- is
modeled by a hydrated pyrophosphate complex
Ca(H 2 O) m 2+(HOPO 2 OPO 2 CH 3 )2- with methyl group
31
Am. J. Biotechnol. Mol. Sci., 2011, 1(1): 30-38
instead of adenosine monophosphate residue.
Calcium ion is supposed to be added to oxygen
atoms of pyrophosphate anion as shown in Chart 1
and donates two coordination bonds. The other four
coordination bonds may be used for addition of the m
water molecules; m being dispersed in the range
from 0 to 4.
(2) These values, however, are firmly dependent on
n, that is on the number of water molecules in
hydrate shell of Ca(H 2 O) n 2+ and Ca(H 2 O) n +.
(3) We may talk about a “trigger”, threshold value of n
(n* = 4), which determines the essential link between
two energetic regimes of reaction 1. At n > 4, it is
endoergic and energy forbidden, while at n < 4 it is
allowed by energy and generates the ion-radical pair.
For reaction 2, the threshold is far larger (n* >4)
because this reaction is significantly more exoergic
(by 4eV). These statements are identical to those
ones derived from calculations for similar reactions of
the ADP-magnesium complexes (Buchachenko et al.,
2010). This itself is a good enough reason for an
attempt to search for the calcium-related magnetic
isotope effect expressed in the bivalent metal
dependent reactions of ATP synthesis directed by
enzymes belonging to the known kinases superfamily.
The value m = 4 corresponds to a completely filled up
six-coordinated shell of Ca2+ ion. Apart of the
complexes 1, we have also calculated the structure
/energy patterns of the ion-radicals 1a (Chart 1)
generated from 1 by electron detachment. (Figure 1)
shows a few typical structures for some selected
values of m. The withdrawal of electron from
complexes 1 results in redistribution of the electron
density and slightly changes the inter-atomic
distances in complexes 1a with respect to those in 1
(see Figure 1).
Taking into account that the CK catalytic site may
contain all ADP molecules, protonated and
deprotonated ones, we have calculated the structure
and
energy
patterns
of
the
complexes
2+
3(they
simulate
Ca(H 2 O) m (OPO 2 OPO 3 СН 3 )
Ca2+(ADP)3- ) and of those ones with the detached
electrons. The structure and energy parameters of
ions Ca(H 2 O) n 2+ and Ca(H 2 O) n + were also calculated
as a function of n, the number of water molecules in
the coordination sphere of calcium ion; n is supposed
to be placed within the range 0 - 6.
Calcium Isotope Effect:
The yields of ATP
synthesized by 40Ca-CK and 43Ca-CK, respectively,
are shown in Figure 3 as a function of CaCl 2
concentration. The ATP yields increases as the
concentration of CaCl 2 goes up reaching a maximum
at [CaCl 2 ]>120 mM with a following decrease inside a
highest calcium concentration range. At low
concentration of CaCl 2 , there is almost no difference
in the ATP yields. However, at [CaCl 2 ] ≥40 mM ,
43
Ca-CK produces ATP more efficiently compared to
40
Ca-CK. At a maximum point ([CaCl 2 ]120 mM), the
isotope effect seen from the ratio of the yields is
equal to 1.8±0.2. Taking into account that the content
of 43Ca in 43Ca-CK is 86.7% only, it is easy to
estimate a net. Referring to 100% of 43Ca, the
resulting isotope effect magnitude is equal to 2.0±0.3.
Energy of Electron Transfer Reactions: The total
energy values of reactions 1 and 2, which simulates
reactions 3 and 4, were determined as a difference
between the total energies of reactants and products.
The reaction is exoergic if the difference is positive.
Vice versa, the reaction is endoergic and its starting
energy is forbidden if the difference is negative. This
approach has an obvious advantage since it helps to
avoid
errors
compensating
some
possible
inaccuracies in energies estimated for individual
reagents and products. Both reactions presented (1
and 2) are to generate the ion-radical pairs, a
“source” of the magnetic isotope effect.
As seen from Figure 4, the Calcium-43 related
magnetic isotope effect is no doubt detectable by
monitoring over the CK-directed creatine production
rate as well. This leads to the very same, listed
above, conclusion derived from the data presented in
(Figure 3).
DISCUSSION
The energies E of reactions 1 and 2 are presented in
Figure 2 as the functions of m and n. This would lead
to the following statements.
It should be noticed first that Ca2+ ion affects the ATP
synthesis with almost the same efficiency as Mg2+
does. (Buchachenko et al., 2008) Likewise, both Mg2+
and Ca2+ ions do not promote any isotope effect
within a low concentration range where the so called
“classical”, i.e. nucleophylic, mechanism of ATP
synthesis dominates.
(1) The energy values are almost independent on m
(m = 0-4), i.e. on the number of water molecules in
the first coordination sphere of complexes
Ca(H 2 O)2+ m (HOPO 2 OPO 3 CH 3 )2− and, therefore,
Ca2+(ADP)2-.
32
Am. J. Biotechnol. Mol. Sci., 2011, 1(1): 30-38
However, some critically big amount of Ca2+ ions
turns on another mechanism of enzymatic
phosphorylation which relies upon a very specific
physical phenomenon, namely the nuclear spin
selective formation of ion-radical pairs. That’s the
Calcium-43 related magnetic isotope effect is about.
formation of ATP and the final ion-radical pair which,
in turn, generates creatine and regenerates Ca2+ by
the reverse electron transfer. It should be pointed out
that both Ca2+ and Ca+ ions are hydrated ones (see
below).
The rate of an ATP synthesis associated with the
singlet channel (reactions 1-3 in Scheme 1) is
suppressed by a spin-allowed reverse electron
transfer in the primary ion-radical pair; this is to
regenerate the starting (initial) reactants and surely
decreases the resulting ATP yield. However, the
43
Ca2+ induced hyperfine coupling of an unpaired
electron with the 43Ca nucleus in Ca+ ion provokes a
singlet-triplet spin conversion inside the primary ionradical pair transforming it into the triplet state where
the reverse electron transfer is spin forbidden.
Moreover, this peculiar effect mentioned looks even
more productive compared to the well-known
nucleophylic reaction path.
All above listed regularities are based on the
following postulates supported by both experiments
and DFT calculations:
(1)
Phosphorylation starts as an electron transfer
reaction which generates an ion-radical pair
comprised of the hydrated Ca+ radical-cation and the
phosphate radical-anion of ADP as the partners;
A triplet phosphorylation channel, therefore, provides
an additional yield of ATP increasing its total
production up to about twice. It should be outlined
that the reaction 1 (Scheme 1) is indeed the electron
transfer path, from Ca2+(ADP)2- or Ca2+(ADP)3complexes to the Ca(H 2 O) n 2+ ion and occurs only at
n ≤ n*. At least two important consequences seem to
derive from this result.
(2)
Due to the singlet-triplet spin conversion that
had happen inside an ion-radical pair, a resulting
chemical reactivity of either triplet or singlet states of
the pair is essentially different one from another. This
is a cause for a remarkably higher ATP yield level
provided by the triplet engaging phosphorylation
channel;
First, the ion-radical mechanism of ATP synthesis is
found to be launched exclusively in the presence of
the excess of Ca2+ ions. This is about to happen on a
condition that the phosphocreatine-free and/or ADPfree Ca(H 2 O) n 2+ ions appear in the CK catalytic site.
(3)
The relative contribution of these spin
channels into the final ATP yield depends on the
electron-nuclear (hyperfine) magnetic coupling of
unpaired electrons with magnetic nucleus 43Ca in the
hydrated Ca+ ion and with 31P in the radical
pyrophosphate fragment of ADP. This Coulomb
coupling induces a singlet-triplet spin conversion and
hence determines the nuclear spin selectivity of the
whole ATP synthesis process.
Secondly, this may explain why as a matter of fact
the ATP synthesis occurs in such specific devices as
the molecular enzymatic machines. The bottom line
is that their functioning includes a compression of
reactants and hence the partial “squeezing” of the
water molecules out of the catalytic site. This is to
dehydrate the Ca2+ ion which helps to activate the
further attachment of electron to Ca(H 2 O) n 2+ ion,
when (if) n ≤ n*. This is a reason why the ATP
synthesis does not occur in water where Сa2+ ions,
as well as Mg2+ ions, (Buchachenko et al., 2010) are
highly hydrated (n >> 4). Compression of reactants in
catalytic site stimulates both nucleophylic and ionradical reactions. However, the mechanisms of this
stimulation are different.
These critical points of the Calcium-43 involving spin
selective biochemical pathway are summarized in the
(Scheme 1). This type of mechanism has been
proposed earlier for the Mg-CK (Buchachenko et al.,
2008) directed ATP synthesis.
The very first reaction step shown in the Scheme 1
implies a transfer of electron from the terminal
phosphate group of ADP to the Ca2+ ion; it generates
a primary ion-radical pair composed of the radicalcation Ca+ and the oxy-radical of ADP (reaction 1 in
(Scheme 1)); all this takes place within a singlet spin
state prior to the forthcoming spin conservation act.
Since the information on a role of calcium as the
physiologically valid agent is certainly important for its
variable medicinal applications, it is hardly possible to
exaggerate a significance of research revealing such
novel aspects of this element’s biological activity as
its enzyme affecting magnetic isotope effects.
The next step is the phosphorylation itself proceeded
as an attack of P=O chemical bond of creatine
phosphate by the ADP oxy-radical (reaction 2). The
resulting oxy-radical decomposes via the -scission
of PO chemical bond (reaction 3) followed by a
33
AMERICAN JOURNAL BIOTECHNOLOGY AND MOLECULAR SCIENCES
ISSN Print: 2159-3698, ISSN Online: 2159-3701, doi:10.5251/ajbms.2011.1.1.30.38
© 2011, ScienceHuβ, http://www.scihub.org/AJBMS
FIGURES
Graphical Abstract (if needed)
Am. J. Biotechnol. Mol. Sci., 2011, 1(1): 30-38
1
1a
2+
Ca(H2O)
-
O
2+
Ca(H2O)
m
O-
O
O
O
2+
O-
HO P O P OCH3
HO P O P OCH3
O
m
2-
2+
Ca(H2O) m (HOPO2OPO3CH3)
O
-
Ca(H2O) m (HOPO2 OPO3 CH3 )
Chart 1. Calcium phosphate complexes.
Fig 1. Schematic presentation of the calcium pyrophosphate complexes. Calcium atoms are gold, phosphorus is
green, oxygen is red, carbon is yellow, and hydrogen is blue. The figures are inter-atomic distances in Å. Structures
for m = 0, 2, 4 are shown in Supporting Information.
35
Am. J. Biotechnol. Mol. Sci., 2011, 1(1): 30-38
E, eV
9,0
8,0
7,0
6,0
5,0
4,0
3,0
2
2,0
1,0
n
0,0
-1,0
0
1
2
3
4
5
6
1
-2,0
Fig 2. Energies of reactions 1 and 2 as a function of the number of water molecules n in the Ca (H 2 O)2+ n ions for
different m, the number of water molecules in Ca(H 2 O) m 2+(HOPO 2 OPO 3 СН 3 )2- (curve 1) and
32+
Ca(H 2 O) m (OPO 2 OPO 3 СH 3 ) (curve 2).
Ax10
50
3
40
2
30
20
1
10
0
0
40
80
120
160
200
[CaCl2], mM
Fig 3. The 40Ca-CK (1) - and 43Ca-CK (2) – directed ATP synthesis rates.Ordinate - A, (c.p.m. γ - [ 32P] ATP/mg CK)
x 103.
36
Am. J. Biotechnol. Mol. Sci., 2011, 1(1): 30-38
Y
35
30
25
20
2
15
10
1
5
0
0
50
100
150
200
[CaCl2 ], mM
Fig 4. The yield of creatine ( Y,
concentration.
M/mg CK) produced by 40Ca-CK (1) and 43Ca-CK (2) as a function of CaCl2
2+
Ca
CH3
O
O O
-O P O AMP
HO2C CH2 N C NH P
+NH
2
O
O
1 40,42,44Ca
.
Ca+
O O
NH
.
O
P
. +
S
O
43Ca
P
O O
O
O
2'
.
Ca+
.
S
O
O.
P
NH
.
O
P
O.
3
.
O
O O
O
NH Ca+
T
+
Ca
O
P
NH
P
O
2
O O
O
.
HN P
O
O
T
Ca
P
O
3'
.
S
.
NH Ca+
+ ATP
T
+ ATP
SCHEME 1. Ion-radical mechanism of the calcium impacted ATP synthesis catalysed by CK.
37
Am. J. Biotechnol. Mol. Sci., 2011, 1(1): 30-38
kinase posphorylation. Proc. Natl. Acad. Sci. U.S.A.,
102, 10793 - 10796.
ACKNOWLEDGEMENTS:
This work was financially supported by Russian
Ministry of Science and Education (Grant NS-2010.3)
and by Russian Foundation for Basic Research
(Grant 08-03-00141). The European INTAS-06/09
Magnetic Field Biological Effects Research Grant is
also acknowledged.
Buchachenko, A. L. (2009) Magnetic Isotope Effect in
Chemistry and Biochemistry; Nova Science Publishers:
New York.
Buchachenko, A. L., Kuznetsov, D. A., Breslavskaya, N. N.
and Orlova, M. A. (2008) Magnesium isotope effects in
enzymatic phosphorylation. J. Phys. Chem. B., 112,
708 - 719.
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