A zinc(II)-based receptor for ATP binding and hydrolysis

Article abstract of DOI:10.1039/b502229k

A protonated Zn(II) complex with a terpyridine-containing pentaamine macrocycle catalyses ATP hydrolysis in the presence of a second metal ion, which acts as cofactor assisting the phosphoryl transfer from ATP to an amine group of the receptor. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b502229k

View Article Online / Journal Homepage / Table of Contents for this issue
COMMUNICATION
www.rsc.org/chemcomm | ChemComm
A zinc(II)-based receptor for ATP binding and hydrolysis{
a
Carla Bazzicalupi, Andrea Bencini,* Antonio Bianchi,* Andrea Danesi, Claudia Giorgi, Carlos Lodeiro,
a
a
a
a
b
b
Fernando Pina, Samuele Santarelli and Barbara Valtancoli
a
a
Received (in Cambridge, UK) 14th February 2005, Accepted 14th March 2005
First published as an Advance Article on the web 24th March 2005
DOI: 10.1039/b502229k
A protonated Zn(II) complex with a terpyridine-containing
pentaamine macrocycle catalyses ATP hydrolysis in the
presence of a second metal ion, which acts as cofactor assisting
the phosphoryl transfer from ATP to an amine group of the
receptor.
binding and can easily protonate in aqueous solutions to give
15
2+x
[ZnLH
x
]
complexes (Scheme 1).
Alternatively, the polyamine chain can be used to bind a second
Zn(II) ion, affording dimetallic complexes. The monometallic L
complexes are in principle promising binding and/or hydrolytic
agents for ATP, since they combine within the same receptor the
peculiar features of both metal-based and polyammonium ATP
binders. Actually, potentiometric measurements{ show that ATP
is strongly bound to the monometallic complexes with L.
ATP plays a basic role in the bioenergetics of all living organisms,
the center for chemical energy storage and transfer being its
triphosphate chain. In Nature ATP hydrolysis is catalyzed by
ATPases; a divalent metal cation, generally Mg(II) or Ca(II), is
involved in the catalytic mechanism, acting as binding site for ATP
As shown in Fig. 1, ATP binding at alkaline pHs affords the
32
ternary complexes [ZnL(OH)ATP]
22
and [ZnLATP] , while
1–3
ATP complexes with protonated species of the Zn(II) complex,
x22
or as cofactor, favoring the phosphoryl transfer process.
However, ATPases which hydrolyze ATP only in the presence of
31
[
x
ZnLH ATP] , prevail at neutral and acidic pHs. A P NMR
4
titration§ shows that the resonances of both the Pc and Pb
‘
‘softer’’ metal cations (Zn(II), Cd(II) or Pb(II)) are also known. In
recent years synthetic hydrolytic systems, from macrocyclic
phosphate groups shift downfield upon coordination of ATP to
+
2+
5–9
polyammonium cations
10–14
ligands,
the [ZnLOH] and [ZnL] complexes, probably due to the
simultaneous involvement of both phosphate moieties in coordina-
to metal complexes with simple
have been developed to elucidate the mechanism of
tion to Zn(II) (Fig. 1).
ATP cleavage. Metal complexes generally cleave phosphate
monoesters and ATP through activation of the phosphate chain
via coordination to the metal center(s) and attack of a metal-
bound hydroxide on phosphorus. Partially protonated polyamine
x22
The formation of the [ZnLH ATP]
complexes, instead, gives
signal with
respect to the corresponding signal of ‘‘free ATP’’, indicating that
in the protonated ternary complexes the P phosphate is involved
x
rise only to a further marked downfield shift of the P
c
5–9
c
macrocycles, instead, can hydrolyze ATP
via binding and
in salt bridges with the positively charged ammonium groups of
the ligand and leading us to propose the interaction mode between
ATP and the protonated Zn(II) complexes sketched in Scheme 2
activation of ATP thanks to the formation of salt bridges between
the phosphate chain and the charged ammonium groups and
subsequent nucleophilic attack of an amine group at the
c-phosphorus of ATP to give a labile phosphoramidate inter-
mediate. In this case it was observed that the presence of Zn(II)
7
reduces the hydrolytic ability of the receptor.
We have recently reported the synthesis of the terpyridine-
1
5
containing macrocycle L. In its monometallic Zn(II) complex the
metal ion is bound to the terpyridine nitrogens, while the
polyamine chain is not involved or weakly involved in metal
Scheme 1
{
Electronic supplementary information (ESI) available: experimental
31
details for the potentiometric and P NMR experiments. Formation
constants of the ATP adducts with L and with its mono- and dimetallic
Zn(II) complexes, plots of time-dependence of ATP concentrations in the
Fig. 1 Distribution diagram of the ternary complexes formed by the
31
Zn(II), L and ATP (1 : 1 : 1 molar ratio) and P NMR chemical shift of
2+
the signals of the P
in the absence of the Zn(II) complex with L as a function of pH. The
chemical shift of the P signal does not change in the presence of the Zn(II)
c b
and P phosphate groups of ATP in the presence and
presence of the Zn–L complex and Zn at various pH values, plot of the
2+
k
OBS values vs. Zn concentration. See http://www.rsc.org/suppdata/cc/b5/
b502229k/
andrea.bencini@unifi.it (Andrea Bencini)
a
*
complex with L and it has been omitted.
2
630 | Chem. Commun., 2005, 2630–2632
This journal is ß The Royal Society of Chemistry 2005

View Article Online
1
(
drawing a). At the same time, H NMR titrations display an
upfield shift of the signals of the adenine protons upon ATP
complexation, as generally observed in the case of p-stacking
interaction of adenine with an aromatic moiety. The protonated
x+2
[
ZnLH
x
]
complexes, therefore, behave as multifunctional
receptors for ATP, thanks to the simultaneous presence of
metal–donors bonds, electrostatic and hydrogen bonding interac-
tions and p-stacking pairing. Actually, the potentiometric study of
this system shows that the Zn(II) complexes with L display a
markedly higher affinity for ATP than the protonated ligand in the
absence of Zn(II), confirming the crucial role of Zn(II) in ATP
binding. Ligand L and its monometallic Zn(II) complexes,
however, do not show any significant ability in ATP hydrolysis.
Addition of an equiv. of Zn(II) ion to solutions containing the
monometallic ternary complexes with ATP leads to the formation
of dimetallic Zn(II) complexes with the nucleotide (Fig. 2); the
second metal is coordinated to the pentaamine chain and therefore
this process competes with protonation of the aliphatic amine
groups. In consequence, monometallic protonated complexes
prevail at acidic pH values even in the presence of a second metal
ion, while dimetallic complexes are formed from neutral to alkaline
pHs. Once again, the dimetallic complexes do not give ATP
hydrolysis. On the contrary, a fast cleavage process is observed by
Fig. 2 Distribution diagram for the system Zn(II)/L/ATP in 2 : 1 : 1
__
molar ratio ( , left y axis) and experimental pseudo-first order rate
constants ($, right y axis) for ATP cleavage at 298.1 K (I 5 0.1,
2
+
22
ZnL] 5 [Zn ] 5 1.65 ? 10 M).
[
with a 1 : 1 molar ratio between the Zn(II) complex and ‘‘free’’
Zn(II)), compared with other polyammonium macrocycles able to
5–9
hydrolyze ATP.
The hydrolytic process is partially inhibited by ADP coordina-
tion (for instance only 70% ATP is cleaved at pH 4), although the
stability of the ADP ternary complexes is lower than that of the
corresponding ATP complexes. Our complex, however, behaves as
a real catalyst, with a turnover number of 13 at pH 4 (298.1 K,
31
2+
P NMR in the acidic pH region,§ where the [ZnLH
4
ATP]
2+
23
22
[
ZnL] 5 [Zn ] 5 1 ? 10 M, [ATP] 5 2 ? 10 M).
complex and free Zn(II) are simultaneously present (Fig. 2).
3
1
Experiments carried out on solutions containing the mono-
metallic Zn(II) complex with L at acidic pHs and increasing
amount of Zn(II) also showed the formation of increasing
percentages of phosphoramidate intermediate (Fig. 3).
The analysis of the time dependence of the P NMR spectra
recorded at 298.1 K shows that the hydrolytic process leads first to
the formation of ADP and a phosphoramidate intermediate (PN),
characterized by the typical signal at 10.06 ppm, which is then fast
hydrolysed to hydrogen-phosphate (P). As shown in Fig. 2, the
rate constants for the process ATP A PN + ADP fit the
The rate of ATP dephosphorylation to give PN and ADP
displays a pseudo first-order dependence on ‘‘free’’ Zn(II)
concentration, giving rise to an overall second-order kinetics
2
+
distribution curve of the tetraprotonated [ZnLH ATP] species,
4
2
2
+
2+
2
21
(
4
v 5 k[ZnLH ATP ][Zn ]), with a rate constant of 4.2 ¡
with a maximum at pH 4 (kOBS 5 (3.2 ¡ 0.2) ? 10 min ), thus
indicating that this complex is indeed the active species. The
hydrolysis rate is among the highest observed for ATP depho-
21
21
.2 M min at 298.1 K and pH 5 4.
0
These data are in accord with a mechanism involving first
32
6
–10
coordination of ATPH (ATP is mainly in its monoprotonated
5+
sphorylation promoted by polyammonium receptors.
Further
3+
complex protonation to give the [ZnLH ATP] species leads to a
5
form at pH 4) to the [ZnLH
3
]
complex and then metal-assisted
complete quenching of the cleavage process. At pH 4, the
phosphoramidate intermediate is formed, together with ADP, in
the first few minutes up to a relatively high percentage (ca. 30%
nucleophilic attack of an unprotonated amine function to give the
PN intermediate (c in Scheme 2).
The fact that ATP cleavage takes place only in the presence of a
‘‘second’’ Zn(II) ion with second order kinetics suggests that the
transition state could be stabilized by this metal ion (b in Scheme 2),
probably through coordination of the metal to the unprotonated
Fig. 3 Percentages of PN intermediate formed in the presence of
2+
increasing amounts of [Zn ] at pH 4 and T 5 298 K. The relative
Scheme 2
concentration is based on the initial concentration of ATP.
This journal is ß The Royal Society of Chemistry 2005
Chem. Commun., 2005, 2630–2632 | 2631

View Article Online
of the zinc complex with L was measured by an initial slope method
monitoring the decrease of the ATP resonances. Potassium hydrogen
phthalate (pH 2.7–4.5), MES (pH 4.5–6.5) and MOPS (pH 6.5–8.5) buffers
were used (50 mM). In a typical experiment, ATP, the zinc complexes with
amine groups of the macrocycle and to the c-phosphate of ATP,
leading to a higher activation of the c-phosphorus to the
nucleophilic attack. At the same time, the PN intermediate could
be stabilized thanks to coordination to the metal, accounting for
the observed relatively high percentage of PN formed during the
cleavage process. In such a way, the second Zn(II) ion could
2 2
L and ZnCl were mixed in D O solution at the appropriate pH and stored
at 298.1 ¡ 0.1 K. The reaction mixture was sampled by drawing a 0.8 ml
portion of solution generally every 2–5 minutes. To quench the hydrolytic
effect, each sample was immediately refrigerated at 278 K and its pH was
adjusted to 7 (in these conditions ATP hydrolysis is negligible for several
‘
‘assist’’ the transfer of the c-phosphate from ATP to an amine
group of the macrocycle. The quenching effect observed with the
2
hours). No ATP hydrolysis was observed in the absence of added ZnCl .
3
1
3+
P NMR spectra were then recorded for each sample, determining the
percentages of each species in solution.
formation of the [ZnL(ATP)H5] complex can be reasonably
attributed to protonation of an amine group of the macrocycle,
which results in no more being available for the nucleophilic
attack.
1
2
H. Dugas, Bioorganic Chemistry: a Chemical Approach to Enzyme
Action, Springer, New York, 1996.
J. E. Estes and P. J. Higgins, (Eds), Actin: Biophysics, Biochemistry and
Cell Biology, Plenum Press, New York, 1994.
W. K u¨ hlbrandt, Nature, 2004, 5, 282–295.
Finally, the formed ADP is replaced by ATP in the ternary
complex, thanks to the higher stability of the ATP complexes with
respect to the ADP ones.
3
4
(a) R. Sharma, C. Rensing, B. P. Rosen and B. Mitra, J. Biol. Chem.,
2000, 275, 3873–3878; (b) Z. Hou and B. Mitra, J. Biol. Chem., 2003,
The present system, therefore, represents a unique case of ATP
dephosphorylation promoted by the simultaneous action of a
metal complex, which is used essentially for substrate anchoring,
and of a second metal, which probably acts as cofactor, assisting
the phosphoryl transfer from ATP to an amine group of the
receptor.
278, 28455–28461.
5
6
M. W. Hosseini, J. M. Lehn, L. Maggiora, K. B. Mertes and
M. P. Mertes, J. Am. Chem. Soc., 1987, 109, 537–544.
M. W. Hosseini, Bioorg. Chem. Front., 1993, 3, 67–112.
7 P. G. Yohannes, K. E. Plute, M. P. Mertes and K. Mertes, Inorg.
Chem., 1987, 26, 1751–1755; K. B. Mertes and M. P. Mertes, Acc.
Chem. Res., 1990, 23, 413–418.
8
G. Papoyan, K. Gu, J. Wiorkiewicz-Kuzcera, K. Kuzcera and
K. Bowman-James, J. Am. Chem. Soc., 1996, 118, 1354–1364.
a
Carla Bazzicalupi, Andrea Bencini,* Antonio Bianchi,*
a
a
a
a
b
Andrea Danesi, Claudia Giorgi, Carlos Lodeiro, Fernando Pina,
b
a
Samuele Santarelli and Barbara Valtancoli
Department of Chemistry, University of Florence, Via della Lastruccia
a
9 A. Bencini, A. Bianchi, E. Garcia-Espa n˜ a, E. C. Scott, L. Morales,
B. Wang, T. Deffo, F. Takusagawa, M. P. Mertes, K. B. Mertes and
P. Paoletti, Bioorg. Chem., 1992, 20, 8–29; A. Andres, J. Arago,
A. Bencini, A. Bianchi, A. Domenech, V. Fusi, E. Garcia-Espa n˜ a,
P. Paoletti and J. A. Ramirez, Inorg. Chem., 1993, 32, 3418–3424.
a
3
, 50019-Sesto Fiorentino, Firenze, Italy. E-mail: andrea.bencini@unifi.it
b
REQUIMTE/CQFB, Departamento de Qu ´ı mica, Universidade Nova de
Lisboa, 2829-516 Monte de Caparica, Portugal.
E-mail: fjp@dq.fct.unl.pt
1
1
0 V. Scheller-Krattiger and H. Sigel, Inorg. Chem., 1986, 25, 2628–2634.
1 N. H. Williams, A.-M. Lebuis and J. Chin, J. Am. Chem. Soc., 1999,
21, 3341–3348.
2 N. H. Williams, J. Am. Chem. Soc., 2000, 122, 12023–12024.
3 J. L. Sessler, K. L. Ross, G. W. Hemmi, W. C. Dow, D. A. Smith,
1
1
1
Notes and references
{
The formation constants of ATP with L and its complexes were obtained
by means of potentiometric measurements in 0.1 M Me NCl at 298.1 K by
using the method and procedure described in reference 9.
V. A. Kral, B. Iverson, T. Mody, M. Darren and R. A. Miller, PCT Int.
Appl. WO 9429316, 1994.
14 Y. Guo, Q. Ge, H. Lin, H. K. Lin, S. Zhu and C. Zhou, Biophys.
Chem., 2003, 103, 119–131.
15 C. Bazzicalupi, A. Bencini, E. Berni, A. Bianchi, A. Danesi, C. Giorgi,
B. Valtancoli, C. Lodeiro, J. C. Lima, F. Pina and M. A. Bernardo,
Inorg. Chem., 2004, 43, 5134–5144.
4
31
§
P NMR experiments were carried out on a 400 MHz instrument at
98 K. In the P NMR titrations, HCl and NMe OH were used to adjust
31
2
the pH values. The pH was calculated from the measured pD values by
4
using the eqn: pH 5 pD 2 0.40. The rate of ATP cleavage in the presence
2
632 | Chem. Commun., 2005, 2630–2632
This journal is ß The Royal Society of Chemistry 2005