Recognition and catalytic hydrolysis of adenosine 5′-triphosphate by cadmium(II) and L-glutamic acid

Article abstract of DOI:10.1080/00958972.2011.608162

Interactions among Cd²⁺, glutamic acid (Glu), and adenosine 5′-triphosphate (ATP) have been studied by potentiometric pH titration, IR, Raman, fluorescence, and NMR methods. In the Cd²⁺-ATP binary system, the main interaction sites are the α-, β-, and γ-phosphate groups, N-1, and/or N-7. Cd²⁺ binds to the N-1 site at relatively low pH and binds to the N-7 site of adenosine ring of ATP with increasing pH. In the Cd²⁺-Glu-ATP ternary system, ATP mainly binds to Cd²⁺ by the triphosphate chain. Oxygens of Glu coordinate with Cd²⁺ to form a complex to catalyze ATP hydrolysis. Hydrolysis of ATP catalyzed by the CdGlu complex was studied at pH 7.0 and 80°C by ³¹P-NMR spectrometry. Kinetics studies showed that the rate constant of ATP hydrolysis was 0.0199 min-1 in the ternary system, which is 9.9-fold faster than that in the ATP solution (2.01 10^-3 min-1). Hydrolysis occurs through an addition-elimination reaction mechanism with Cd²⁺ regulating the recognition and catalytic hydrolysis of ATP; water participates in the hydrolysis reaction of ATP at different steps with different functions in the ternary system.

Full text of DOI:10.1080/00958972.2011.608162

Journal of Coordination Chemistry
ISSN: 0095-8972 (Print) 1029-0389 (Online) Journal homepage: http://www.tandfonline.com/loi/gcoo20
Recognition and catalytic hydrolysis of adenosine
5′-triphosphate by cadmium(II) and L-glutamic acid
Jin-Ying Zhou & Gong-Xuan Lu
To cite this article: Jin-Ying Zhou & Gong-Xuan Lu (2011) Recognition and catalytic hydrolysis
of adenosine 5′-triphosphate by cadmium(II) and L-glutamic acid, Journal of Coordination
Chemistry, 64:20, 3441-3453, DOI: 10.1080/00958972.2011.608162
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Date: 01 June 2016, At: 13:30

Journal of Coordination Chemistry
Vol. 64, No. 20, 20 October 2011, 3441–3453
Recognition and catalytic hydrolysis of adenosine
59-triphosphate by cadmium(II) and L-glutamic acid
JIN-YING ZHOUyz and GONG-XUAN LU*y
yState Key Laboratory for Oxo Synthesis and Selective Oxidation, Lanzhou Institute of
Chemical Physics, Chinese Academy of Sciences, Lanzhou, Gansu 730000, China
zGraduate University of Chinese Academy of Sciences, Beijing 100049, China
(Received 23 December 2010; in final form 18 July 2011)
Interactions among Cd2þ, glutamic acid (Glu), and adenosine 50-triphosphate (ATP) have been
studied by potentiometric pH titration, IR, Raman, fluorescence, and NMR methods. In the
Cd2þ–ATP binary system, the main interaction sites are the ꢀ-, ꢁ-, and ꢂ-phosphate groups,
N-1, and/or N-7. Cd2þ binds to the N-1 site at relatively low pH and binds to the N-7 site of
adenosine ring of ATP with increasing pH. In the Cd2þ–Glu–ATP ternary system, ATP mainly
binds to Cd2þ by the triphosphate chain. Oxygens of Glu coordinate with Cd2þ to form a
complex to catalyze ATP hydrolysis. Hydrolysis of ATP catalyzed by the CdGlu complex was
studied at pH 7.0 and 80ꢀC by 31P-NMR spectrometry. Kinetics studies showed that the rate
constant of ATP hydrolysis was 0.0199 minꢁ1 in the ternary system, which is 9.9-fold faster
than that in the ATP solution (2.01 ꢂ 10ꢁ3 minꢁ1). Hydrolysis occurs through an addition–
elimination reaction mechanism with Cd2þ regulating the recognition and catalytic hydrolysis
of ATP; water participates in the hydrolysis reaction of ATP at different steps with different
functions in the ternary system.
Keywords: ATP; Cd2þ; L-Glutamic acid; Recognition; Catalytic hydrolysis
1. Introduction
Adenosine 50-triphosphate is an important enzymatic compound and plays a funda-
mental role in bioenergetics of all living organisms by transferring its triphosphate chain
[1, 2]. ATP hydrolysis is catalyzed by both ATPase and a divalent metal ion (Mg2þ
,
Cd2þ, etc.), which provides a binding site for ATP or serves as a cofactor that catalyzes
the phosphoryl transfer process [3–8]. Because of the complicated nature of the
biological system, the roles of ATPase and metal ions in the catalytic hydrolysis of ATP
at the molecular level remain unknown. Therefore, non-enzymatic catalysis of ATP
hydrolysis has attracted attention from both chemists and biologists. Although studies
have been widely conducted on metal ions’ impacts on ATP hydrolysis [8–11], the
interactions between cadmium ions and ATP have been described only in a few studies
[12–14] and the mechanisms for catalytic ATP hydrolysis involving cadmium ions have
not been elucidated due to its known biotoxic effects [15, 16]. However, cadmium ions,
*Corresponding author. Email: gxlu@licp.cas.cn
Journal of Coordination Chemistry
ISSN 0095-8972 print/ISSN 1029-0389 online ß 2011 Taylor & Francis
http://dx.doi.org/10.1080/00958972.2011.608162

3442
J.-Y. Zhou and G.-X. Lu
which are as effective in activation of ATPase as magnesium ions [17], are involved in
certain biological processes [18, 19] and can fully replace magnesium ions as metal
cofactors for ATP hydrolysis. Thus, studying the structural organization and functional
activity of ATPase in vivo is conducive to understanding the roles of cadmium ions in
the catalytic hydrolysis of ATP.
ATP hydrolysis can be catalyzed not only by metal ions, but also other bioligands, such
as amino acids or polyamines. Both natural polyamines and synthetic ligands, e.g.,
macrocyclic polyammonium, phenanthroline, and polyoxomolybdates, have been
developed to elucidate mechanisms for reactions involving ATP and metal ions
[8, 20–22]. However, there are only a few previous studies focusing on the roles of
metal ions and amino acids in recognition and catalytic hydrolysis of ATP [23, 24].
Structural modifications or mutations of any of the amino acid residues could affect the
functions of the living cells. For example, enzymes might lose catalytic activities when key
amino acid residues are chemically modified or mutated to other amino acid residues.
L-Glutamic acid, an important component in biological systems, has been found in the
binding region of ATPase and plays an important role in ATP hydrolysis in
living organisms [25, 26]. The ATPase activity decreases when the Glu residue
is mutated to another amino acid residue in the ATPase pocket [27–29]. It is important
to understand how Glu and a metal ion recognize ATP and then catalyze ATP hydrolysis.
In this article, we present the results of the interactions among ATP, Glu, and Cd2þ to
elucidate the reaction mechanism of ATP hydrolysis catalyzed by Cd2þ and Glu.
2. Experimental
2.1. Materials
Adenosine 50-triphosphate disodium salt (99%), adenosine-50-diphosphate (ADP, 99%),
adenosine-50-monophosphate (AMP, 99.7%), and L (þ)-glutamic acid (99%) were pur-
chased from Acros Organics (USA). CdCl2 ꢃ 2.5H2O was purchased from the Shanghai
Hengxin Chemical Reagent Co. (China). Deuterium oxide (D2O, 99.9%) was obtained
from Cambridge Isotope Laboratories Inc. All these chemicals were used without
purification. The structures of ATP and L-Glutamic acid are shown in scheme 1.
2.2. Potentiometric pH titration
A solution of ATP, metal ion, and/or secondary ligand glutamic acid in a molar ratio of
1 : 1 or 1 : 1 : 1 was titrated with a NaOH solution at 25 ꢄ 0.1ꢀC under an argon
atmosphere. Constant ionic strength was maintained with 0.1 mol Lꢁ1 NaNO3 and a
total volume of 25 mL used in each titration. The calibration of the composite electrode
was checked using standard buffer solutions with pH values of 4.00 and 9.18. The
calculations were carried out by SCMAR program [30] (Newton–Gauss nonlinear least-
squares) on an IBM compatible computer.
2.3. Fluorescent, IR, and Raman measurements
Fluorescence spectra were recorded on a F-7000 spectrofluorophotometer (Hitachi,
Japan), using 5/5 nm slit widths, with an excitation wavelength of 281 nm and the

Hydrolysis of adenosine 50-triphosphate
3443
Scheme 1. Structures of ATP and L-glutamic acid.
emission was read from 300 to 550 nm. Solution (3.0 mL) containing an appropriate
concentration of ATP, ADP, and AMP was titrated by successive additions of Cd2þ or
Glu to generate a final concentration of 0–1.0 ꢂ 10ꢁ3 mol Lꢁ1 of Cd2þ and
0–1.0 ꢂ 10ꢁ3 mol Lꢁ1 of Glu at pH 3.0. IR and Raman spectra were measured with a
Nicolet Nexus 670 FT–IR spectrometer (America) and a Nicolet 950 FT–Raman
spectrometer (America), respectively.
2.4. NMR measurements
1H- and 31P-NMR spectra were recorded on an Inova 400 MHz spectrometer.
31P-NMR chemical shifts were referenced to 85% phosphoric acid. The pH of the
solution was adjusted to the desired value using 0.1 mol Lꢁ1 NaOH or HCl at room
temperature.
2.5. Kinetics
Kinetic studies were performed at 80ꢀC by following the time-dependent change in the
integrals from the resolved 31P-NMR signals for Tꢀ, Tꢁ, and Tꢂ of ATP, Dꢀ and Dꢁ for
ADP, the peak OP for the inorganic phosphate, and PN for intermediate species.
For NMR measurements, solution (0.5 mL 10% D2O) containing ATP with and
without Cd2þ and/or glutamic acid was placed in a 5 mm NMR tube and all the
concentrations of ATP, Glu, and Cd2þ were 0.0363 mol Lꢁ1
.
3. Results and discussion
3.1. Potentiometric pH titration
Table 1 presents the overall stability constants (log ꢁ) of the molecular complexes
appearing in the ATP/Cd2þ/Glu binary and ternary systems, determined from
computer analysis of the potentiometric titration data. The species distribution
diagrams of the complexes are shown in figure 1.

3444
J.-Y. Zhou and G.-X. Lu
Table 1. Overall stability constants (log ꢁ) for the complex of ATP with Cd2þ
or Glu and ATP with Cd2þ and Glu.
Species
log ꢁ
Species
log ꢁ
20.2777
24.8169
HATP
6.5891 ꢄ 0.07
10.2955 ꢄ 0.06
9.6455
13.9818
16.15
5.188 ꢄ 0.0001
8.3371 ꢄ 0.0002
13.2887
H2GluATP
H3GluATP
H4GluATP
CdGluATPOH
HCdGluATP
H2CdGluATP
H3CdGluATP
H4CdGluATP
H2ATP
HGlu
H2Glu
27.9636
13.0958 ꢄ 0.0056
22.2123 ꢄ 0.0057
27.1415 ꢄ 0.0057
31.2567 ꢄ 0.0058
34.3496 ꢄ 0.0059
H3Glu
CdATP
HCdATP
HGluATP
Figure 1. Species percentage distribution diagrams for the binary and ternary systems: (a) the ATP–Cd2þ
binary system and (b) the ATP–Cd2þ–Glu ternary system.
Stability constants of ATP and ATP–Cd2þ complexes, listed in table 1, are consistent
with literature values [12, 31]. Figure 1(a) shows the distribution of the various forms
reported for an ATP–Cd2þ solution. HCdATP was dominant in the pH range of 2–6
and up to a maximum at pH 4.0. The CdATP species forms when pH42.5 with
maximum concentration at pH 7.0, the ideal pH level for bioactivity in the biological
systems. The complex H4GluATP appeared at pH below 5.0. H3GluATP, H2GluATP,
and HGluATP were dominant in the pH ranges 2.0–7.0, 2.0–10.0, and 4.0–12.0,
respectively, and reached a maximum at pH 4.0, 6.0, and 8.0, respectively (figure 1 of
Supplementary Material). In the ternary ATP–Cd2þ–Glu system, the following
protonated complexes were found (figure 1b): H4CdGluATP, H3CdGluATP,
H2CdGluATP, and HCdGluATP. H4CdGluATP species was already observed to
form at pH below 2.0 and the other complexes, H3CdGluATP, H2CdGluATP, and
HCdGluATP, formed stepwise as pH increased, and reached a maximum at pH 3.5, 4.5,
and 7.0, respectively. All the species are predominant in the pH range and their
maximum amounts were higher than 60%. HCdGluATP got to its maximum
concentration (490%) at pH 7.0, which is the ideal pH for bioactivity.

Hydrolysis of adenosine 50-triphosphate
3445
Figure 2. Changes in fluorescence spectra of ATP with gradual addition of Cd2þ in an aqueous solution
(pH 3.0): [ATP] ¼ 1.0 ꢂ 10ꢁ4 mol Lꢁ1, [Cd2þ] ¼ 0–1.0 ꢂ 10ꢁ3 mol Lꢁ1; excitation wavelength: 281 nm.
3.2. Fluorescence
Fluorescent character of ATP only exists in a narrow wavelength range in aqueous
solution. ATP emits light in the 310–500 nm range when the excitation wavelength is
281 nm. ADP showed less fluorescence intensity and AMP displayed a very weak signal
compared with that of ATP in the same conditions (figure 2 of Supplementary
Material). Adenine showed a very weak emission from 310 to 500 nm with completely
different characteristics than those for AMP, ADP, and ATP. Adenosine almost has no
fluorescence compared with that of nucleotide [32]. The number of phosphates in the
nucleotide is able to shift the light emission and modify its intensity. Quenched spectra
can be used to study the molecular environment in the vicinity of the chromophore
molecule [32–34].
Fluorescence emission spectra of 1 ꢂ 10ꢁ4 mol Lꢁ1 ATP with and without Cd2þ in
aqueous solutions at pH 3.0 are shown in figure 2. Obviously, ATP emitted a strong
fluorescence band at 384 nm when the excitation wavelength was 281 nm. Intensities of
the emission peaks of ATP gradually decrease as Cd2þ is added into the ATP solution.
Chemical quenching is ruled out as the cause of weakening fluorescence intensity of
ATP after addition of Cd2þ because Cd2þ did not compete for the excitation photon,
since Cd2þ does not absorb in the spectral range (300–550 nm). Dilution quenching is
also ruled out because the volume and concentration of ATP were kept constant. Thus,
it is reasonable to suggest that cadmium ions bound to oxygen of phosphate of ATP
formed a CdATP complex and thus modified the fluorescence intensity of ATP.
Further research on the interactions between ATP and Cd2þ have been carried out by
comparing fluorescence intensities of ATP, ADP, and AMP containing aqueous
solutions with addition of Cd2þ (figure 3). Fluorescence intensities of ATP and ADP
decrease with the addition of Cd2þ, while those of AMP were nearly unchanged. These
results indicate that Cd2þ mainly binds ꢁ- and ꢂ-phosphate groups while interactions
between the ꢀ-phosphate of ATP and Cd2þ were very weak, which will be confirmed by
NMR spectra in section 3.4. In figure 3 of the Supplementary Material, the fluorescence
intensities of nucleotide (ATP, ADP, and AMP) were nearly unchanged with addition

3446
J.-Y. Zhou and G.-X. Lu
Figure 3. Relative fluorescence intensity of ATP, ADP, and AMP obtained by titration with Cd2þ
;
[ATP] ¼ [ADP] ¼ [AMP] ¼ 1.0 ꢂ 10ꢁ4 mol Lꢁ1, [Cd2þ] from 0 to 1.0 ꢂ 10ꢁ3 mol Lꢁ1
.
Table 2. Characteristic IR bands (cmꢁ1) of the spectra of the complexes in the different ATP–Cd2þ–Glu
systems and free ATP.
Compound
N–H
C–N(N-7)
ꢃ
assPO(ꢀ and ꢁ)
P–O–C
ꢃsPO(ꢂ)
P–O–P
ATP
1648
1641
1640
1495
1512
1513
1230
1221
1249
1050
1081
1079
968
992
992
902
920
913
ATP þ Cd2þ
ATP þ Cd2þ þ Glu
of Glu into the nucleotide–Cd2þ solution, suggesting that the phosphate groups of
nucleotide mainly interact with Cd2þ and the interactions between the phosphate
groups of nucleotide and Glu were very weak in the ternary systems.
3.3. IR and Raman spectra
IR and Raman spectra of ATP and its complexes are shown in figures 4 and 5 of the
Supplementary Material. The wavenumbers and positions [35, 36] are summarized in
table 2. Small shifts in the bands, observed from 4000 to 1700 cmꢁ1, may be due to
rearrangement of the strong hydrogen bonding network of free ATP upon complex-
ation [37]. The absorption at 1495 cmꢁ1 assigned to the purine ring vibration of ATP
showed a shift of about 15 cmꢁ1 for its complexes, suggesting N-7 binding [37].
The band at 968 cmꢁ1 assigned to the symmetric stretch of the ꢂ-phosphate in free ATP
[36–38] shifted to 992 cmꢁ1 and its intensity was greatly reduced upon complexation. In
all cases, IR spectra showed a shift of ca 24 cmꢁ1 for these absorptions. The intense
band at 1249 cmꢁ1, attributed to asymmetric stretch of the ꢀ- and ꢁ-phosphates of free
ATP [36, 38], shifted to 1220–1230 cmꢁ1. Thus, oxygens of ꢀ-, ꢁ-, and ꢂ-phosphates of
ATP participate in coordination for all compounds.
The Raman spectrum of ATP between 600 and 1600 cmꢁ1 is important for
analytical studies. The main features of the Raman spectra of ATP (figure 5 of

Hydrolysis of adenosine 50-triphosphate
3447
Supplementary Material) are in general agreement with previous reports [39]. Most
observed bands can be assigned to vibrations of phosphate and adenine, which
predominantly interacted with metal ions [40–42]. Detectable variations were found in
the phosphate vibration region at 1121 cmꢁ1 where a shift of 11 cmꢁ1 was observed in
the ATP–Cd2þ binary system and a shift of 3 cmꢁ1 in the ATP–Cd2þ–Glu ternary
system. Ring vibrations of the five-membered ring at 1498 cmꢁ1 shifted and their
intensities were greatly reduced in both the ATP–Cd2þ binary and ATP–Cd2þ–Glu
ternary systems. The intensity reductions at 1309 cmꢁ1 and the line broadening at
724 cmꢁ1 indicate that the N-7 site of the adenine ring was the site of interaction
between ATP and Cd2þ and/or Glu, since these two vibrations can be attributed to
either five- or six-membered rings. Thus, the triphosphate groups and the adenine ring
engage in ATP’s interactions with Cd2þ and/or Glu in the ATP–Cd2þ binary system
and ATP–Cd2þ–Glu ternary system, respectively.
3.4. NMR
3.4.1. ATP–Cd2þ binary system. Interactions between Cd2þ and ATP were investi-
gated by 1H- and 31P-NMR spectrometry. The change in 1H chemical shift as a function
of pH is in agreement with that reported by Wang et al. [43]. Figure 6 of the
Supplementary Material shows the normal proton NMR spectrum of the adenine ring
of ATP, which serves as the reference for other spectra. The two peaks at 8.008 and
8.337 ppm were assigned to H-2 and H-8 protons of ATP at pH 7.0, respectively. The
upfield shifts of H-2 in the pH range 3–5 reflect proton ionization at the N-1 site [43].
The signals of H-2 and H-8 shift downfield at pH ꢅ 6, indicating that the terminal
phosphate group and the N-1 site of ATP were deprotonated [43].
The chemical shifts of H-2 and H-8 of the adenine ring of ATP changed significantly
in the ATP–Cd2þ binary system compared with those in the ATP solution (figure 6 of
Supplementary Material). Two conformations, the so-called syn (the base and sugar are
on the same side with respect to the glycosidic bond) and anti-conformers [44], exist in
the ATP molecule. At relatively low pH, ATP takes a syn conformation, while it takes
an anti-conformer when pH45 [45]. As shown in figure 6 of the Supplementary
Material, the chemical shifts of H-2 were downfield in the binary system when pH ꢆ 3.
It is well known that coordination of a metal ion to a binding site deshields neighboring
protons and the resonances of such protons are shifted downfield [43, 46, 47]. Protons,
however, are added to the N-1 site to form zwitterion species þH2(ATP)3ꢁ, with the N-1
site being positively charged when pH ꢆ 3. Compared with Hþ, Cd2þ has the advantage
of smaller charges. Thus, Cd2þ can at least partially replace the proton to bind to the
N-1 site at low pH [48] and the resonance signals of H-2 were shifted downfield
compared with those in free ATP solution. However, with increasing pH, the chemical
shifts of H-2 were upfield, indicating that Cd2þ does not interact with the N-1 site of
ATP at pH43 because ATP becomes an anti-conformer and no geometric advantage
exists between the N-1 site of ATP and Cd2þ. The corresponding chemical shifts of H-2
were upfield, probably due to the known Cd2þ promoted self-association of ATP [49].
Figure 6 of the Supplementary Material shows that the chemical shifts of H-8 were
downfield in the whole pH range, due to Cd2þ directly coordinating with the N-7 site of
adenine ring of ATP, which was also supported by previous studies [12, 48–50].

3448
J.-Y. Zhou and G.-X. Lu
The ATP molecule can interact with metal ions not only by nitrogens of the purine
nucleus, but also by oxygens of the triphosphate chain. In 31P-NMR spectra of Cd2þ
–
ATP binary system (figure 7 of Supplementary Material), the chemical shifts of all the
phosphorus atoms changed with the addition of Cd2þ into ATP solution, which,
together with the results of the fluorescence, IR, and Raman studies, indicate that all
phosphate groups participated in this reaction. Compared with the 31P chemical
shifts of all the phosphate groups in free ATP solution, the change of signals of ꢁ- and
ꢂ-phosphates was much larger than that of the ꢀ-phosphate of ATP in the ATP–Cd2þ
binary system. Cd2þ mainly interacted with ꢁ- and ꢂ-phosphates and the interac-
tion between Cd2þ and ꢀ-phosphate was weaker than that among Cd2þ, ꢁ-, and
ꢂ-phosphate groups in the whole pH range. The results are consistent with the findings
from the molecular modeling calculations (figure 8 of Supplementary Material).
3.4.2. ATP–Cd2þ–Glu ternary system. The complexation of CdATP in the presence of
Glu has been studied by 1H- and 31P-NMR spectrometry (figures 9 and 10 of
Supplementary Material). The purpose was to find out whether an ATP–Cd2þ–Glu
complex can be observed.
Addition of Glu into the ATP solution caused downfield shifts of the signals of H-2
and H-8 in the whole pH range (figure 9 of Supplementary Material). The N-1 site is a
positive reaction center because N-1 is protonated when pH55 and the carboxyl group
of Glu is a negative center of the interaction because it has a negative charge in the
whole pH range. Therefore, the carboxyl of Glu interacted with the N-1 site of ATP by
hydrogen bond and electrostatic interactions [51]. With increasing pH, N-1 is
deprotonated, and an inverse interaction takes place with N-1 of ATP becoming a
negative center and the protonated amine of Glu taking part in the interaction as a
positive center. Intermolecular interactions between the carboxyl of Glu and ATP are
impossible because there are no positive centers in the ATP molecule at relatively high
pH. At the same time, the phosphate group of ATP also engaged in noncovalent
interactions with Glu, supported by changes in the position of the 31P-NMR signals.
Compared with the 31P chemical shifts of all the phosphate groups in free ATP solution,
the presence of Glu resulted in downfield shifts of both ꢁ-P (0.04 ppm) and ꢂ-P
(0.15 ppm) and no shift occurred in ꢀ-P at pH 7.0. The results indicate that the terminal
phosphate group is the primary active site of ATP with Glu by electrostatic interaction
and hydrogen bonding [52], although the interaction between Glu and the triphosphate
groups of ATP is very weak. Compared with those in the ATP solution, the 31P signals
of ꢀ-, ꢁ-, and ꢂ-P of ATP in the ternary system were downfield shifted by 0.125, 2.33,
and 1.69 ppm, respectively, and those in the Cd2þ–ATP binary system by 0.21, 2.45, and
1.75 ppm, respectively, at pH 7.0 (figure 10 of Supplementary Material). Cd2þ could
coordinate with the oxygen of the carboxyl groups of Glu [53], leading to decreased
activity of Cd2þ with the phosphate groups of ATP. Since the positive charge density of
Cd2þ bonded to Glu, it decreased the electron-withdrawing effect of Cd2þ to oxygens of
phosphate groups of ATP [53]. Thus, competition exists between ATP and Glu in
binding to Cd2þ in the ternary Cd2þ–ATP–Glu system. One interesting aspect of 31P
signals for the ternary system is that the chemical shifts for all three phosphorus atoms
of ATP were almost the same as those in the binary Cd2þ–ATP system, indicating that
the major interaction of the phosphate groups of ATP was with cadmium in the ternary
Cd2þ–ATP–Glu system.

Hydrolysis of adenosine 50-triphosphate
3449
3.4.3. Catalytic hydrolysis of ATP. Kinetic measurements of ATP hydrolysis were
carried out by the following time-dependent changes of the integrals from the resolved
31P-NMR signals of ꢀ-, ꢁ-, and ꢂ-P of ATP and peaks of inorganic phosphates.
The ATP hydrolysis catalyzed by Cd2þ and Glu was studied at 80ꢀC and pH 7.0 using
31P-NMR. The 31P signals of ATP at the different hydrolysis times are illustrated
in figure 4. ATP hydrolyzed to ADP and phosphate (OP), and then yielded AMP
and OP. The observed rate constant kobs (equation (1)) was obtained from the plot of
ln([ATP]/[ATP]0) as a function of time (figure 5), where [ATP]0 and [ATP] are the initial
and actual concentrations of ATP, respectively.
R ¼ kobs½ATPꢇ ¼ ꢁd½ATPꢇ=dt
ð1Þ
From the kinetic study, Cd2þ and Cd2þ–Glu accelerate the reaction of ATP
hydrolysis moderately with rate constants of 1.84 ꢂ 10ꢁ2 minꢁ1 and 1.99 ꢂ 10ꢁ2 minꢁ1
,
Figure 4. 31P-NMR spectra of ATP hydrolysis at different reaction times in the ternary system.
Concentrations of Cd2þ, ATP, and Glu are 0.0363 mol Lꢁ1, D2O/H2O ¼ 1 : 9 at 80ꢀC and pH ¼ 7.0.
(a) t ¼ 0 h, (b) t ¼ 0.5 h, and (c) t ¼ 2 h. Tꢀ, Tꢁ, and Tꢂ for the ꢀ-, ꢁ- and ꢂ-phosphorus of ATP; Dꢀ and Dꢁ for
ADP; M for AMP and OP for inorganic phosphate.

3450
J.-Y. Zhou and G.-X. Lu
Figure 5. Rate constants for ATP hydrolysis in different systems. ATP (g), Cd2þ–ATP (ꢈ), and Cd2þ
ATP–Glu (m).
–
respectively, at pH 7.0 (figure 5), which were about 9.15- and 9.9-fold faster than in the
ATP solution (2.01 ꢂ 10ꢁ3 minꢁ1), respectively. Cd2þ not only directly interacts with the
N-7 site and coordinates with ꢀ-, ꢁ- and ꢂ-P of ATP but also coordinates with water to
form a complex [53], which is a good nucleophile to attack the terminal phosphate of
ATP. Thus, the catalysis of cadmium ions is greater than that of calcium and
magnesium ions [11, 54, 55]. For efficient catalytic hydrolysis of ATP, strong binding of
the substrate by the catalyst is required. The binding of ATP by the Glu-metal complex
has the following dual effects on the rate acceleration: (a) neutralization of the negative
charge of the substrate and (b) an increase in the susceptibility of the phosphorus center
of ATP [56].
All these experimental results allowed us to find the reaction mechanism for catalysis
of ATP hydrolysis by Cd2þ and Glu in the ternary Cd2þ–ATP–Glu system. Catalytic
efficiency depends on the structural requirements of the complex itself, which involves a
protonation pattern, electrostatic interaction, hydrogen bonding effects, and spatial
arrangement of the reacting species at the catalytic site [57]. According to an addition–
elimination mechanism [58], we propose that ATP hydrolysis was catalyzed by Cd2þ
and Glu in the ATP–Cd2þ–Glu ternary system, as shown in scheme 2. The hydrolysis of
ATP has been divided into three steps. The first step is recognition and binding among
ATP, Cd2þ, and Glu, as discussed in previous sections. The second step involves an
intermolecular nucleophilic attack on ꢂ-P of ATP by HCdGlu-H2O, the formation of
an intermediate, and the release of ADP. The cadmium binds to the ꢀ-, ꢁ-, and
ꢂ-phosphate groups of ATP and the oxygen of the carboxyl groups of Glu. CdGlu-H2O
acts as a nucleophile to attack the terminal phosphate in ATP–Cd2þ–Glu ternary
system to form a phosphorylated intermediate. The phosphorylated intermediate was
found in 31P-NMR spectra in other ATP-metal ion-amino acid ternary systems [55].
However, the intermediate has not been observed in the Cd2þ–Glu–ATP ternary system
since formation of the phosporamidate intermediate is related to pH, reaction time,
ATP concentration, etc. The final step of the mechanism at neutral pH involves the
capture of a water and the coordination bond CdGlu-PO3(OH) cleavages to give

Hydrolysis of adenosine 50-triphosphate
3451
Scheme 2. Postulated mechanism for ATP hydrolysis catalyzed by Cd2þ–Glu.
HPO24ꢁ and regenerate the catalyst. The hydrolysis of ATP in the ternary system is
faster than that of free ATP, from a combination of the stronger nucleophile and the
electrostatic catalysis.
4. Conclusions
Both the N-1 and/or N-7 of the adenosine ring and all the triphosphate groups of ATP
are reaction sites in the Cd2þ–ATP binary system. At relatively low pH, Cd2þ can
partially or fully replace the proton to interact with N-1 site of adenosine; the efficiency
of the interactions of N-1 with Cd2þ decreases with increasing pH as N-1 is
deprotonated. Cd2þ directly interacts with the N-7 site of the adenosine ring in the
whole pH range and promotes self-association of ATP at relatively high pH. Cd2þ
coordinates with the ꢀ-, ꢁ-, and ꢂ-phosphate groups of ATP in the whole pH
range, although the interactions between ꢀ-phosphate and Cd2þ are weaker than those
of ꢁ- and ꢂ-phosphate groups. In the Cd2þ–Glu–ATP ternary system, ATP mainly
binds to the cadmium ion. Interactions between ATP and Glu are very weak, negligible
compared with that between ATP and Cd2þ. The oxygens of the carboxyl groups of Glu
coordinate with Cd2þ to form a complex to catalyze ATP hydrolysis.
The hydrolysis of ATP catalyzed by Cd2þ and Glu in the Cd2þ–Glu–ATP ternary
system has been investigated by 31P-NMR spectra. Cd2þ and Cd2þ–Glu accelerate the
rate of ATP hydrolysis and the roles of Cd2þ–Glu in the ternary system are analyzed.
The possible mechanism of ATP hydrolysis catalyzed by Cd2þ–Glu has been proposed
on the basis of an addition–elimination reaction mechanism. The results show that
metal ions can regulate the hydrolysis reaction of ATP and water participates in the

3452
J.-Y. Zhou and G.-X. Lu
ATP hydrolysis process at different steps with different functions. This is useful
to obtain more information about the key amino acid residues and metal ions that
serve as cofactors in the ATPase effect on the ATP synthesis and hydrolysis at the
molecular level.
Acknowledgments
The authors thank Mr Zhiqiang Shen for his technical assistance in obtaining NMR
spectra. This study is supported by the 973 Program under the Grant no. 2009CB22003.
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