JOURNAL OFInorganicBiochemistry

Journal of Inorganic Biochemistry 98 (2004) 917–924

www.elsevier.com/locate/jinorgbio

Catalysis of phosphoryl transfer from adenosine-50-triphosphate(ATP) by trinuclear ‘‘chelate’’ complexes

Ruiguang Ge a,1, Hai Lin b, Xinhe Xu a, Xuesong Sun c,1, Huakuan Lin a,*,Shourong Zhu a, Baofeng Ji a, Fenghua Li a, Hongxing Wu a

a Department of Chemistry, Nankai University, Tianjin 300071, PR Chinab State Key Laboratory of Functional Polymer Materials for Absorption and Separation, Nankai University, Tianjin 300071, PR China

c Department of Microbiology, Nankai University, Tianjin 300071, PR China

Received 9 January 2004; received in revised form 11 March 2004; accepted 23 March 2004

Available online 22 April 2004

Abstract

The chelate ligand 2,9-di(60-a-phenol-n-20,50-diazahexyl)-1,10-phenanthroline (L) was synthesized and fully characterized. This

ligand formed six protonated species in the solution. The bindings of the ligand to the nucleotide anions ATP, ADP and AMP were

described in detail, with equilibrium constants given for each species formed. The strength of binding increased with the number of

protons, corresponding to an increase in the number of hydrogen bonds and an increase in the coulombic attractive forces. At the

same time, the coordination properties of the ternary complexes formed from the chelate ligand above, M (M¼Zn2þ, Cd2þ) andadenosine-50-triphosphate (ATP) were studied. The metal complexes of the chelate recognize the nucleotides via multiple interac-

tions similar to those occurring in the center of enzymes. The hydrolysis of ATP was studied with the mononuclear and trinuclear

chelate complexes.

� 2004 Elsevier Inc. All rights reserved.

Keywords: Chelate complex; Nucleotides; Ternary complex; Cd(II)

1. Introduction

It is now well-known that metal ions are essential in

various biological processes, including those with nu-cleic acids and their derivatives [1–4]. Many enzymes

(and ribozymes) require one or more metal ions as a

cofactor in catalyzing phosphate ester hydrolysis and

transesterfication. Notably, hydrolysis of adenosine-50-triphosphate (ATP) occurs via highly efficient metal-

loenzymatic reactions catalyzed by the ATPases and

plays a key role in numerous processes: photosynthesis

phosphorylation (chloroplast ATPase), oxidative phos-phorylation (mitochondrial ATPase), muscle action

(myosin ATPase), etc. However, despite a large knowl-

* Corresponding author. Fax: +86-22-23502458 (H. Lin).

E-mail address: hklin@nankai.edu.cn (H. Lin).1 Present address: Department of Chemistry, The University of

Hong Kong, Hong Kong, China.

0162-0134/$ - see front matter � 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.jinorgbio.2004.03.007

edge base of metalloenzyme crystal structures, kinetic

and binding data, and extensive studies with model

systems, the detailed role the metal ions play is still

unclear [5–10]. There is therefore considerable interest inanalyzing the controlling factors and the mechanism of

these reactions, as well as discovering non-biological

compounds which might catalyze them [11].

Polyamine ligands have been studied widely because

of their importance in coordination chemistry [12], bi-

omimetic studies [13] and supramolecular catalysis [14].

Successful catalysis by polyamine ligands relies on the

recognition and selectivity of the initially bound sub-strate and the release of the product formed following

chemical transformation. In aqueous solution, the sta-

bility of the recognition complex is related to hydrogen

bonding and coulombic interactions as well as the geo-

metrical ‘fit’ of the substrate with respect to the receptor

polyammonium ligands. A feature desirable for catalytic

behavior is strong binding of the substrate but relatively

918 R. Ge et al. / Journal of Inorganic Biochemistry 98 (2004) 917–924

weak binding of the product so as to favor dissociation

of the resultant complexes and release of the ligands to

continue the catalytic cycle [15].

Current interest focuses on the molecular recognition

of nucleotides by the polyamine ligands and their metalcomplexes [15–17], but the effects of different metal ions

as well as water molecules in the process of catalyzing

ATP hydrolysis have not been studied thoroughly. We

now report the synthesis and the protonation constants

of a chelate ligand 9-di(60-a-phenol-n-20,50-diazahexyl)-1,10-phenanthroline (L) as well as the supramolecular

interactions between the metal ions with nucleotides/L.

The ATP-hydrolysis is followed with 13P-NMR spectra.Such study can lead to a better understanding of certain

biological functions as well as of new catalysts of value

in chemical synthesis.

2. Experimental

2.1. Materials

Most of the starting materials were obtained com-

mercially and were purified prior to use. The sodium salt

of ATP, ADP and AMP were purchased from Aldrich

Chemical Co. The aqueous stock solutions of the nu-

cleotides were freshly prepared. All other materials used

in the experiments including the potassium hydrogen

phthalate, HNO3, KOH, and the metal ion solutions of

CHO

OH

N

N

N

N

N

N N

OHC CHO

1)

2) NaBH4

NH2CH2CH2NH2

Fig. 1. The processes of the

Zn(NO3)2 and Cd(NO3)2 were prepared with redistilled

water. The concentration of KOH used for titration was

established with potassium hydrogen phthalate. The

exact concentrations of the stock Zn(NO3)2 and

Cd(NO3)2 solutions were determined by ethylenedi-aminetetraacetic acid (EDTA) titrations.

2.2. Equipments

Elemental analysis was made on a Perkin–Elmer

240C elemental analyzer. The 1H-NMR, and 31P-NMR

spectra were recorded with a Varian UNITY-plus 400

MHz Spectrometer. IR spectra were obtained as KBrdisks on a Euinox 55 FT Spectrometer (Bruker). Elec-

trospray Ionization Mass Spectrometry (ESI-MS) was

obtained on a Bruker ESQUIRE-LC. Titration was

carried out with a Beckman pH Meter (model U71)equipped with a 39481 combination glass electrode.

2.3. Synthesis of the chelate ligand L (Fig. 1)

2.3.1. Synthesis of 1-a-phenol-n-2,5-diazapentaneEthylenediamine (6 g) was dissolved in ethyl alcohol

(20 ml). Salicylaldehyde (1.22 g) dissolved in ethyl al-

cohol (70 ml) was added dropwise to the above solution

during a period of 10 h with stirring at room tempera-

ture. After the addition was completed, the mixture was

stirred for another 24 h at room temperature. NaBH4

(0.76 g) was added slowly in small quantities and then

OH

NH2

OH

NH NH2

H HN

HO

HHN

HO

NaBH4

L

ligand L preparation.

Table 1

The protonation constants for the chelate ligand L

Equilibria L

[LH]/[L][H] 10.19� 0.05

[LH2]/[LH][H] 9.75� 0.03

[LH3]/[LH2][H] 8.88� 0.05

[LH4]/[LH3][H] 7.54� 0.06

[LH5]/[LH4][H] 5.50� 0.03

[LH6]/[LH5][H] 4.66� 0.02

25.0� 0.1 �C, I ¼ 0:1 M KNO3, [L]¼ 0.5 mM.

R. Ge et al. / Journal of Inorganic Biochemistry 98 (2004) 917–924 919

the mixture was filtrated. In the filtrate concentrated

HCl was added. The deposit was suspended in ethyl

alcohol (100 ml) and was added NaOH. After filtration,

the solvent was removed under reduced pressure, leaving

a white residue, 1-a-phenol-n-2,5-diazapentane (yield60.4%). 1H-NMR (D2O, ppm): d 6.98, 6.61 (s, 4H, Ph); d3.92 (s, 4H, CH2–Ar); d 3.53, 3.40 (s, 8H, N–CH2–CH2–

N). IR (KBr pellet, cm�1): 1161 ðmCNÞ; 3423 ðmNHÞ; 456,865, 1595 (Ar); 2845 (mCHÞ; 3640 ðmOHÞ. Elemental

analysis: Calc. for C9H14N2O, H: 8.49%; C: 65.03%; N:

16.85%. Found H: 8.41%; C: 65.12%; N: 16.74%. ESI-

MS: 166.4 (calculated for C9H14N2O 166.1).

2.3.2. Synthesis of 2,9-di(60-a-phenol-n-20,50-diazahexyl)-1,10-phenanthroline (L)

1-a-phenol-n-2,5-diazapentane (1.66 g) dissolved in

ethanol (50 ml) was stirred at room temperature and was

added 2,9-dicarboxaldehyde-1,10-phenanthroline (1.18

g) in 15 min. The mixture was stirred for another 12 h

and then was added NaBH4 (1.52 g) in small quantities

in an ice–water bath. The solvent was rotator-evapo-rated and 25 ml water was added to the residue. The

aqueous mixture was extracted with chloroform (3� 20

ml). The organic fractions were combined, and then

dried over Na2SO4. The mixture was filtered and chlo-

roform was removed on a rotary evaporator to give

yellow oil which was further dissolved in a least volume

of ethyl alcohol. After adding suitable volume of HCl,

yellowish precipitation was collected, washed with ethylalcohol, recrystallized with ethanol/ether and then dried

in a vacuum desiccator. Yield 50.1%. 1H-NMR (D2O,

ppm): d 8.65, 8.11, 7.89 (s, 6H, Phen); d 7.07, 6.66 (s, 8H,

Ph); d 4.74 (s, 4H, CH2-Phen); d 4.20 (s, 4H CH2–Ar); d3.62, 3.50 (s, 8H, N–CH2–CH2–N). IR (KBr pellet,

cm�1): 1161 (mCN); 3423 ðmNHÞ; 456, 865, 1595 (Ar); 2845

ðmCHÞ; 3640 ðmOHÞ; 1586, 1345, 865 (Phen). Elemental

analysis: Calc. for C32H36N6O2 � 4HCl, H: 5.87%; C:56.30%; N: 12.32%. Found H: 5.93%; C: 56.11%; N:

12.39%. ESI-MS: 537.3 (682.36 for calculated

C32H36N6O2 � 4HCl; the difference is due to the reason

that during the process of obtaining the ESI-MS spectra,

the four molecules of HCl are discharged).

2.4. Determination of equilibrium constants by potentio-

metric titrations

Potentiometric determination was measured in a 50

ml jacketed cell thermostated at 25.0� 0.1 �C by a re-

frigerated circulating water bath. Anaerobic conditions

were maintained using pre-purified N2 as an inert at-

mosphere, and the ionic strengths were maintained by

adding KNO3 to achieve I ¼ 0.1 M. The calibration of

the glass electrode was the same as described in the lit-erature [18]. In a typical experiment, the ligand L was

dissolved in an adequate amount of dilute HNO3 and

then titrated with 0.1 M NaOH. The values of

Kx ¼ 1:008� 10�14, cþH ¼ 0:825 of water were used for

the calculation. The calculations were carried out by

TITFIT, a Newton–Gauss–Marquardt nonlinear least-

squares program [19]. The final results are the averages

of three independent titrations, each titration containingabout 50 experimental points.

2.5. Kinetics

Kinetic studies were performed by following the time

evolution of the 31P-NMR spectra. Since 31P-NMR

signals of ATP, ADP, AMP and orthophosphate (OP)

are distinct, ATP-hydrolysis can be monitored conve-niently and accurately by following the changes in the

concentrations of various species [20]. Eight-five percent

H3PO4 was used as an external standard. In a typical

experiment, a 0.6 ml solution (10% D2O/H2O) in a 5 mm

tube containing 3.3 mM ATP and 3.3 mM L and/or

corresponding concentrations of metal ions was ad-

justed to the desired pH at 70 �C.

3. Results and discussion

3.1. Protonation of the ligand

The decimal logarithms of stepwise protonation

constants of the ligand L are listed in Table 1. The

species percentage distribution diagram of L is shown inFig. 2.

Although the ligand L consists of eight aza donors, it

has only six stepwise protonation constants. The two

nitrogen atoms of phen (phenanthroline) are not pro-

tonated in the pH range studied (2–10.5), due to the

lower pKHH-phen ¼ 4.75 [21] vs. 9–11 of secondary nitrogen

atoms, as well as the electron-withdrawing effects of the

already protonated ammoniums on the electron densityof phen. The six protonation constants listed for the li-

gand vary from 1010:19 to 104:66, depending on how

many protons are present. By comparison with the

values of ethylenediamine (pK1 ¼ 7:08, pK2 ¼ 9:89) andortho-cresol (pK ¼ 10:26) [22], the first two protonation

constants should belong to the phenyl group. Due to the

larger electron-withdrawing effect of p-phen than p-benzene, the amino groups neighboring with phen have

Table 3

The binary stability constants of the ligand L with respective M2þ

(Zn2þor Cd2þ) in the absence or presence of ATP

M2þ:L:At Equlibria Zn2þ Cd2þ

(a)

1:1:0 LgbH4ML 41.53 40.58

LgbH3ML 32.57 31.29

LgbH2ML 23.05 17.85

3:1:0 LgbH4ML 41.53 40.58

LgbH M L 31.64 30.39

2 4 6 80

10

20

30

40

50

60

70

80

90

100

10

H6L

H5L

H4L

H3L

H2L

HL

L

%

pH

Fig. 2. Percent distribution diagram for species formed in the L system

as a function of pH. The charge is omitted. (25.0� 0.1 �C, I ¼ 0:1 M

KNO3, [L]¼ 0.5 mM.)

920 R. Ge et al. / Journal of Inorganic Biochemistry 98 (2004) 917–924

weaker basicity than the ones neighboring with benzene,

so the third and the forth constants should belong to the

amino groups neighboring with benzene and the othertwo constants should belong to the amino groups

neighboring with phen. Owing to the build-up of posi-

tive charge on the chelate ligand and the consequentially

increased coulombic repulsion for an additional proton,

each successive protonation becomes weaker, as re-

flected by a lower protonation constant.

3.2. The stability constants of L with nucleotides

Protonation of the receptors gives charged species,

for example, protonated polyammonium can bind single

inorganic phosphates, such as orthophosphate, pyro-

phosphate and triphosphate, as well as nucleotides, such

as AMP, ADP and ATP in aqueous solution [15]. The

interactions of the protonated polyamine ligand L with

the nucleotides anions have been determined by poten-tiometric equilibrium methods and the binding con-

stants are listed in Table 2.

The data in Table 2 show an increase in the binding

strength with the number of protons on NuL (Nu rep-

Table 2

The stability constants of the ligand with respective ATP, ADP or

AMP

Equlibria ATP ADP AMP

LgbH6NuL 53.71 52.45 46.65

LgbH5NuL 48.96 47.85 42.13

LgbH4NuL 43.96 43.02 36.71

LgbH3NuL 38.57 37.62 30.68

LgbH2NuL 32.21 31.57 24.79

LgbHNuL 25.76 25.11

Nu represents ATP, ADP, or AMP. 25� 0.1 �C, I ¼ 0:1 M KNO3,

[L]¼ [Nu]¼ 0.5 mM.

resents ATP, ADP, or AMP) increased to a maximum of

six (corresponding to the maximum number of hydrogen

bonds between the host and guest). The number of the

H-bonds of adducts is equal to or higher than

the number of protons present in the complexes, sincethe H-bonds can form not only in the form of NH � � �O,

but also via NH � � �N, OH � � �O and OH � � �N.

The magnitudes of the binding strength between the

chelate ligand and the substrates ATP, ADP and AMP

decrease in the order ATP>ADP>AMP, as one would

expect from the corresponding decreases in the length of

phosphate chain, the number of hydrogen bondings and

coulombic forces. Thus, the recognition of nucleotidesby the chelate ligand via multiple interactions relies on

the length of phosphate chain and the charge of sub-

strates. According to the stability constants shown in

Table 2, the catalytic conversion of ATP!ADP in the

presence of protonated chelate ligand L is reasonable.

The product of the reaction, ADP, is less strongly bound

than ATP due to its shorter phosphate backbone and

fewer negative charges, which facilitates the catalyticconversion of ATP.

3.3. The stability constants of divalent metal ions with L

The stability constants of divalent metal ions with L

are shown in Table 3(a). The coordination patterns of

the complexes (M2þ:L¼ 3:1 as an example) may be as

follows: In the low pH range, the mode H4ML is formed(in this mode, there are three five-membered chelates

which make the complex stable). With the increase of

pH, the proton in the amino group neighboring with

benzene dissociates. Due to the chelate ring effect, the

2 2

LgbM3L 20.81 19.89

LgbOHM3L 13.77 13.65

(b)

1:1:1 LgbH5MLAt 57.09 55.19

LgbH4MLAt 50.98 49.42

LgbH3MLAt 46.12 45.36

LgbH2MLAt 42.19 37.51

3:1:1 LgbH4MLAt 57.34 56.13

LgbH2M2Lat 54.77 52.61

LgbM3LAt 47.89 46.94

LgbOHM3LAt 41.97 40.25

25� 0.1 �C, I ¼ 0:1 M KNO3, [ATP]¼ [L]¼ 0.5 mM.

R. Ge et al. / Journal of Inorganic Biochemistry 98 (2004) 917–924 921

second metal ion coordinates with the oxygen in one of

the phenyl group and the nitrogen in one of the amino

groups neighboring with benzene and forms H2M2L.

With the further increase of pH, all the protonated

protons are lost and the third metal ion takes part in thecoordination and forms another chelate ring. When pH

increases further, one of the water coordinated with the

metal ion deprotonates to give the monohydroxyl

complex OHM3L which is a good nucleophile in neutral

or slightly basic solution.

From Table 3(a), we find that the stability constants

sequence of Zn(II)>Cd(II) is prevalent all throughout

the coordination patterns. Zn and Cd belong to the XIIgroup of the periodic system of elements and lie in the

forth and fifth period, respectively. From Zn to Cd, the

nuclear charge increases 18. As for Zn(II) and Cd(II),

they have the same effective nuclear charge due to the

same shielding effect of the inner s-, p- and d-electrons

but the radius of Cd(II) is larger than that of Zn(II). As

a result, the corresponding stability constants of Zn(II)-

complexes are higher than those of Cd(II)-complexes.

3.4. Interactions between divalent metal ions and ATP/L

The binding constants of the divalent metal ions with

ATP/L are listed in Table 3(b). For the 3:1:1

(Cd2þ:L:ATP) system, the distribution diagram Fig. 3

shows that there are four ternary species, namely

H4CdLAt, H2Cd2LAt, Cd3LAt and OHCd3LAt (Atrepresents ATP). Complex Cd3LAt is dominant at

around pH 5.5, whereas OHCd3LAt is a major species

at pH> 7.5.

Comparing the binding constants of the M2þ/ATP/L

with the corresponding constants of M2þ/L, consider-able interactions apparently took place when the nu-

cleotide was introduced to the 1:1 and 3:1 (M2þ:L)

3 4 5 6 7 80

10

20

30

40

50

60

H6L

Cd

H4CdLAt

H4Cd

2LAt

Cd3LAt OHCd

3LAt%

pH

Fig. 3. Percent distribution diagram for species formed in 3:1:1

(Cd2þ:L:ATP) system as a function of pH. The charge is omitted.

(25� 0.1 �C, I ¼ 0:1 M KNO3, [ATP]¼ [L]¼ 0.5 mM, [Cd]¼ 1.5 mM.)

systems. The nucleotides can offer additional coordina-

tion sites to metal ion and thus increase the stability of

the ternary complexes.

We measured the 31P-NMR spectra of ATP, 1:1

(L:ATP) as well as 3:1:1 (Cd2þ:L:ATP) systems at pH8.0. For 1:1 (L:ATP) system, ATP’s three signals (a, b,c; a-P is neighboring with adenosine) show shifts of

about 0.083, 0.461 and 1.917 ppm, respectively, com-

pared with the chemical shifts of ATP alone. At this

experimental condition, HLAt is dominant in the system

with a little amount of H2LAt. The chemical shift upon

the binding of ATP by protonated L is induced mainly

by the electrostatic interactions and the formation ofhydrogen bonds between the negatively charged oxygen

atoms of ATP and the protonated hydrogen on the

phenyl group. By observing the chemical shifts of ATP’s

three signals, c-P is the one to interact with the pro-

tonated phenyl group in HLAt. b-P may also take part

in the interaction in H2LAt. As to 3:1:1 (Cd2þ:L:ATP)

system studied, OHM3LAt is dominant in the system

and ATP’s three signals shift 0.295, 2.156 and 5.284ppm, respectively. This indicates that b- and c-P take

part in the coordination and c-P is most likely the one to

coordinate with two metal ions due to the fact that the

c-signal shifts most. From this, we can deduce the pos-

sible coordination pattern of OHM3LAt (Fig. 4).

The chelate ligand L can incorporate three metal ions

with four secondary nitrogen atoms, two nitrogens in

the phen as well as two phenyl groups. The metal ionsalso bind with the substrate. Such bindings, involving

coordination bonds and hydrogen bonds as well as

electrostatic interactions, can bind the nucleotide anions

quite strongly. The metal ions can greatly activate the

phosphate linkage for the nuleophilic attack: the doubly

charged terminal –OPcO2�3 group may be coordinated to

the two divalent metal ions (bonded to the phenyl group

N

N

NH NH

NH NH

PO

P

O

P-O

O-

-OO-

M

M

M

O-

O-

O

-OO-

OH

AdO

Fig. 4. The possible binding structure of OHM3LAt. Coordinated

water is not given.

922 R. Ge et al. / Journal of Inorganic Biochemistry 98 (2004) 917–924

and the amino group neighboring with benzene), as a

result the electrons of the phosphate are strongly with-

drawn by the metal ions; furthermore, the orbitals of the

phosphate are mixed with the orbitals of metal ions and

form new hybrid orbitals. According to the potentio-metric titrations, the metal ions release one proton from

their coordinated waters to the aqueous phase. The

metal-ion-bound hydroxide ion is a strong nucleophile,

and what’s more, the phosphate is so activated that the

reaction of hydrolysis can efficiently proceed.

3.5. Hydrolysis of ATP

The hydrolysis of ATP at 70 �C catalyzed by the

protonated L with or without corresponding concen-

trations of metal ions was carried out at I ¼ 0:1 M

KNO3, pH ¼ 8.0 and was followed by 31P-NMR

spectroscopy. The time course of hydrolysis by 3:1:1

(Cd2þ:L:ATP) was shown in Fig. 5. In the experimental

condition studied, the hydrolysis products did not have

any observable inhibitory effect on the hydrolysis ofATP. The observed first-order rate constants kobs are

obtained by linear fit of the plot of logð½ATP�=½ATP�0Þas a function of time Eq. (1), in which [ATP]0 and [ATP]

are the initial concentration and the concentration of

ATP at certain time during the hydrolysis, respectively.

r ¼ kobs½ATP� ¼ �d½ATP�=dt: ð1Þ

Fig. 5. Observation of ATP-hydrolysis by 31P-NMR spectroscopy as a functio

ATP, 9.9 mM Cd2þ and 3.3 mM L at an apparent pH of 8.0 in D2O:H2O 1:9

in ppm relative to external 85% H3PO4; the signals are identified by the follow

Db for ADP; M for AMP; OP for inorganic phosphate. (Ta and Da overlap.

From Table 4, we can see that as to these two

divalent metal ions, the hydrolysis rates of the 3:1:1

(M2þ:L:ATP) modes are higher than those of the

1:1:1 modes (at the experimental pH, OHM3LAt of 3:1:1

modes and H2MLAt of 1:1:1 modes are dominant, al-though in different degrees for different metal ions).

That is understandable. Just as what was discussed in

Section 3.4, OHM3LAt is a strong nucleophile (metal-

bound hydroxide ion) and the phosphate linkage is ac-

tivated so strongly that the nuleophilic attack can easily

take place. As for H2MLAt, the nucleophilic reagent is

the lone pair of electrons on the oxygen. The lone pair of

electrons’ nucleophilic activity is not so strong as that ofthe hydroxide ion. Furthermore, the phosphate linkage

is not so activated as that in OHM3LAt (in the mode of

OHM3LAt, three metal ions interacted with each other

to activate the phosphate linkage; whereas in the mode

of H2MLAt, only one metal ions). Due to the said

reasons, the 3:1:1 (M2þ:L:ATP) modes of complexes

have a higher kobs than the corresponding 1:1:1 modes.

In the 3:1:1 (M2þ:L:ATP) modes, Cd-complex has amuch higher kobs than Zn-complex, which may be due to

the reason that the relative content of OHCd3LAt is

about three times more than the content of OHZn3LAt

at the experimental conditions. The said reason is un-

derstandable when considering the following: The

binding between the metal ion Zn(II) or Cd(II) and L/

ATP increases the electron density of the metal ions in

n of time. Proton-decoupled 31P-NMR spectra (at 81 MHz) of 3.3 mM

at 70 �C recorded at times indicated (in minutes); the chemical shifts are

ing symbols: Ta, Tb, Tc for the a-, b-, c-phosphate groups of ATP; Da,

The reference of the 31P signals downfield shifts for about 0.678 ppm.)

Table 4

The first-order rate constants (kobs � 10�4 min�1) for the hydrolysis of

ATP in the presence or absence of L with/without different metal ions

M2þ:L:ATP M2þ kobs � 10�4 (min�1)

1:1:1 Zn2 7.5

1:1:1 Cd2 8.3

3:1:1 Zn2 8.5

3:1:1 Cd2 138

0:0:1 – 2

0:1:1 – �0a

70� 0.1 �C, I ¼ 0:1 M KNO3, pH 8.0, [ATP]¼ 3.3 mM.aNo hydrolysis was observed during the experimental period of 10 h.

ADP + HPO42-

M M

M

PO

O_

OOP-O

O_

OO

AdO

OH

M

M

POO

O

O OH

HOH

P-O

M

P-O

O_

OO

AdO

P-O

OH

H

Fig. 6. The proposed mechanism of ATP hydroysis by OHM3LAt. L is

not shown.

R. Ge et al. / Journal of Inorganic Biochemistry 98 (2004) 917–924 923

different degrees. Zn(II) forms stronger bonds with L

and ATP than Cd(II) does (Table 3), so the electron

density of Zn(II) increases more than that of Cd(II).

When the effect of the increase of electron density on the

metal ions’ coordination activity to the water outweighs

the effect of the ion size, the water on the Cd(II) is more

liable to release proton. As a result, the Cd-complex has

more nucleophilic reagents. Another possibility is thatthe radius of Cd(II) is larger than that of Zn(II), as a

result the OH� coordinated to Cd(II) may be in a more

favorable place for the nucleophilic attack than the OH�

coordinated to Zn(II).

It is worth noting that after adding equivalent

amount of ligand L to the solution of ATP at pH 8.0,

the rate of hydrolysis kobs dropped from 2� 10�4 to

about 0 min�1. This contrasts with the findings of LehnJ.M. that protonated macrocyclic polyamines can cata-

lyze the hydrolysis of ATP [11,20]. The reasons for the

difference may be as follows. At the pH studied, HLAt is

dominant in the system with a little amount of H2LAt,

as discussed in Section 3.4. The interaction between

ATP and L could not favor ATP for the nuleophilic

attack due to the reasons that the phosphate linkage on

ATP was not activated as discussed for the complex ofOHM3LAt and that the system did not have a strong

nucleophilic reagent. On the other hand, L may protect

ATP from the attack of trace amount of free OH�bysteric effect. As a result, ATP hydrolysis has been in-

hibited by the ligand L.

The proposed mechanism of the hydrolysis of ATP

by OHM3LAt is schematically depicted in Fig. 6. First,

the phosphate linkage is greatly activated as discussed inSection 3.4. Then, the phosphate is attacked by the hy-

droxide ion which is in a suitable position for the nu-

cleophilic attack. The positive charges accumulated in

the metal-hydroxo cluster stabilize the negatively-

charged transition state of ATP hydrolysis (the transi-

tion state is more negatively charged than the initial

state, and is stabilized to a great extent by the adjacent

positive charges). Because of these factors, the penta-coordinated intermediate is efficiently formed. In the

process of the breakdown of the intermediate, the water

coordinated to the metal ions serves as an acid catalyst.

4. Conclusion

One new chelate ligand 2,9-di(60-a-phenol-n-20,50-diazahexyl)-1,10-phenanthroline L was synthesized andfully characterized. The protonation constants of the

ligand as well as the supramolecular interactions be-

tween the metal ions with nucleotides/the chelate ligand

were determined, as a result the possible binding modes

of ATP/L as well as M2þ/ATP/L have been proposed.

The 31P-NMR spectra showed that mainly c-P, some-

times b-P, oxygens took part in the coordination with

the metal ions (M2þ/ATP/L) or the protonated hydro-gen on the phenyl group (ATP/L). On the basis of the

above investigation, the hydrolysis of ATP was carried

out with a satisfactory observed rate constant for tri-

Cd2þ complex and an inhibitory effect on ATP hydro-

lysis has been found for the ligand L. A possible

mechanism of ATP hydrolysis has been proposed, which

showed that a pentacoordinated intermediate may be

formed during the hydrolysis.

Acknowledgements

This project was supported by the National Science

Foundation of PR China (No. 29971018) and the Nat-

ural Science Foundation of Tianjin (No. 023605811).

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