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Catalysis of phosphoryl transfer from adenosine-5′-triphosphate (ATP) by trinuclear “chelate” complexes
JOURNAL OF
Inorganic Biochemistry 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 Shourong Zhu a, Baofeng Ji a, Fenghua Li a, Hongxing Wu a b
a,*
,
a Department of Chemistry, Nankai University, Tianjin 300071, PR China 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 (M ¼ Zn2þ , Cd2þ ) and adenosine-50 -triphosphate (ATP) were studied. The metal complexes of the chelate recognize the nucleotides via multiple interactions similar to those occurring in the center of enzymes. The hydrolysis of ATP was studied with the mononuclear and trinuclear chelate complexes. Ó 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 nucleic 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 metalloenzymatic reactions catalyzed by the ATPases and plays a key role in numerous processes: photosynthesis phosphorylation (chloroplast ATPase), oxidative phosphorylation (mitochondrial ATPase), muscle action (myosin ATPase), etc. However, despite a large knowl*
Corresponding author. Fax: +86-22-23502458 (H. Lin). E-mail address: [email protected] (H. Lin). 1 Present address: Department of Chemistry, The University of Hong Kong, Hong Kong, China. 0162-0134/$ - see front matter Ó 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 in analyzing 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], biomimetic studies [13] and supramolecular catalysis [14]. Successful catalysis by polyamine ligands relies on the recognition and selectivity of the initially bound substrate and the release of the product formed following chemical transformation. In aqueous solution, the stability of the recognition complex is related to hydrogen bonding and coulombic interactions as well as the geometrical ‘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
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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 metal complexes [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 13 P-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.
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 ethylenediaminetetraacetic acid (EDTA) titrations. 2.2. Equipments Elemental analysis was made on a Perkin–Elmer 240C elemental analyzer. The 1 H-NMR, and 31 P-NMR spectra were recorded with a Varian UNITY-plus 400 MHz Spectrometer. IR spectra were obtained as KBr disks on a Euinox 55 FT Spectrometer (Bruker). Electrospray 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. Experimental 2.3. Synthesis of the chelate ligand L (Fig. 1) 2.1. Materials Most of the starting materials were obtained commercially 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 nucleotides were freshly prepared. All other materials used in the experiments including the potassium hydrogen phthalate, HNO3 , KOH, and the metal ion solutions of
2.3.1. Synthesis of 1-a-phenol-n-2,5-diazapentane Ethylenediamine (6 g) was dissolved in ethyl alcohol (20 ml). Salicylaldehyde (1.22 g) dissolved in ethyl alcohol (70 ml) was added dropwise to the above solution during a period of 10 h with stirring at room temperature. 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
N
NH2
NH
CHO
OH
OH
NH2CH2CH2NH2
NH
1) N OHC 2) NaBH4
N
NaBH4
OH
HN
N
HO
N
HO
CHO
H N
NH
NH2
L Fig. 1. The processes of the ligand L preparation.
R. Ge et al. / Journal of Inorganic Biochemistry 98 (2004) 917–924
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 (yield 60.4%). 1 H-NMR (D2 O, ppm): d 6.98, 6.61 (s, 4H, Ph); d 3.92 (s, 4H, CH2 –Ar); d 3.53, 3.40 (s, 8H, N–CH2 –CH2 – N). IR (KBr pellet, cm1 ): 1161 ðmCN Þ; 3423 ðmNH Þ; 456, 865, 1595 (Ar); 2845 (mCH Þ; 3640 ðmOH Þ. Elemental analysis: Calc. for C9 H14 N2 O, H: 8.49%; C: 65.03%; N: 16.85%. Found H: 8.41%; C: 65.12%; N: 16.74%. ESIMS: 166.4 (calculated for C9 H14 N2 O 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-evaporated 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 Na2 SO4 . The mixture was filtered and chloroform 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 ethyl alcohol, recrystallized with ethanol/ether and then dried in a vacuum desiccator. Yield 50.1%. 1 H-NMR (D2 O, 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); d 3.62, 3.50 (s, 8H, N–CH2 –CH2 –N). IR (KBr pellet, cm1 ): 1161 (mCN ); 3423 ðmNH Þ; 456, 865, 1595 (Ar); 2845 ðmCH Þ; 3640 ðmOH Þ; 1586, 1345, 865 (Phen). Elemental analysis: Calc. for C32 H36 N6 O2 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 C32 H36 N6 O2 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 potentiometric titrations Potentiometric determination was measured in a 50 ml jacketed cell thermostated at 25.0 0.1 °C by a refrigerated circulating water bath. Anaerobic conditions were maintained using pre-purified N2 as an inert atmosphere, 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 literature [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
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Kx ¼ 1:008 1014 , cþ H ¼ 0:825 of water were used for the calculation. The calculations were carried out by TITFIT, a Newton–Gauss–Marquardt nonlinear leastsquares program [19]. The final results are the averages of three independent titrations, each titration containing about 50 experimental points. 2.5. Kinetics Kinetic studies were performed by following the time evolution of the 31 P-NMR spectra. Since 31 P-NMR signals of ATP, ADP, AMP and orthophosphate (OP) are distinct, ATP-hydrolysis can be monitored conveniently and accurately by following the changes in the concentrations of various species [20]. Eight-five percent H3 PO4 was used as an external standard. In a typical experiment, a 0.6 ml solution (10% D2 O/H2 O) in a 5 mm tube containing 3.3 mM ATP and 3.3 mM L and/or corresponding concentrations of metal ions was adjusted 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 in Fig. 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 protonated in the pH range studied (2–10.5), due to the lower pK H H-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 density of phen. The six protonation constants listed for the ligand 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) and ortho-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 pbenzene, the amino groups neighboring with phen have Table 1 The protonation constants for the chelate ligand L Equilibria
L
[LH]/[L][H] [LH2 ]/[LH][H] [LH3 ]/[LH2 ][H] [LH4 ]/[LH3 ][H] [LH5 ]/[LH4 ][H] [LH6 ]/[LH5 ][H]
10.19 0.05 9.75 0.03 8.88 0.05 7.54 0.06 5.50 0.03 4.66 0.02
25.0 0.1 °C, I ¼ 0:1 M KNO3 , [L] ¼ 0.5 mM.
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R. Ge et al. / Journal of Inorganic Biochemistry 98 (2004) 917–924 100 90
H4L
H6L
80
H3L
70
H5L
60
H2L
%
50 40
HL
30 20
L
10 0 2
4
6
8
10
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.)
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 other two constants should belong to the amino groups neighboring with phen. Owing to the build-up of positive charge on the chelate ligand and the consequentially increased coulombic repulsion for an additional proton, each successive protonation becomes weaker, as reflected 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, pyrophosphate 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 potentiometric equilibrium methods and the binding constants 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 repTable 2 The stability constants of the ligand with respective ATP, ADP or AMP Equlibria
ATP
ADP
AMP
LgbH6 NuL LgbH5 NuL LgbH4 NuL LgbH3 NuL LgbH2 NuL LgbHNuL
53.71 48.96 43.96 38.57 32.21 25.76
52.45 47.85 43.02 37.62 31.57 25.11
46.65 42.13 36.71 30.68 24.79
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, since the 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 nucleotides by the chelate ligand via multiple interactions relies on the length of phosphate chain and the charge of substrates. 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 catalytic conversion 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 :L ¼ 3:1 as an example) may be as follows: In the low pH range, the mode H4 ML 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 Table 3 The binary stability constants of the ligand L with respective M2þ (Zn2þ or Cd2þ ) in the absence or presence of ATP M2þ :L:At (a) 1:1:0
3:1:0
(b) 1:1:1
3:1:1
Equlibria
Zn2þ
Cd2þ
LgbH4 ML LgbH3 ML LgbH2 ML
41.53 32.57 23.05
40.58 31.29 17.85
LgbH4 ML LgbH2 M2 L LgbM3 L LgbOHM3 L
41.53 31.64 20.81 13.77
40.58 30.39 19.89 13.65
LgbH5 MLAt LgbH4 MLAt LgbH3 MLAt LgbH2 MLAt
57.09 50.98 46.12 42.19
55.19 49.42 45.36 37.51
LgbH4 MLAt LgbH2 M2 Lat LgbM3 LAt LgbOHM3 LAt
57.34 54.77 47.89 41.97
56.13 52.61 46.94 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
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 H2 M2 L. With the further increase of pH, all the protonated protons are lost and the third metal ion takes part in the coordination and forms another chelate ring. When pH increases further, one of the water coordinated with the metal ion deprotonates to give the monohydroxyl complex OHM3 L 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 XII group 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 H4 CdLAt, H2 Cd2 LAt, Cd3 LAt and OHCd3 LAt (At represents ATP). Complex Cd3 LAt is dominant at around pH 5.5, whereas OHCd3 LAt is a major species at pH > 7.5. Comparing the binding constants of the M2þ /ATP/L with the corresponding constants of M2þ /L, considerable interactions apparently took place when the nucleotide was introduced to the 1:1 and 3:1 (M2þ :L)
60
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systems. The nucleotides can offer additional coordination sites to metal ion and thus increase the stability of the ternary complexes. We measured the 31 P-NMR spectra of ATP, 1:1 (L:ATP) as well as 3:1:1 (Cd2þ :L:ATP) systems at pH 8.0. 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, compared with the chemical shifts of ATP alone. At this experimental condition, HLAt is dominant in the system with a little amount of H2 LAt. The chemical shift upon the binding of ATP by protonated L is induced mainly by the electrostatic interactions and the formation of hydrogen 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 protonated phenyl group in HLAt. b-P may also take part in the interaction in H2 LAt. As to 3:1:1 (Cd2þ :L:ATP) system studied, OHM3 LAt is dominant in the system and ATP’s three signals shift 0.295, 2.156 and 5.284 ppm, 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 possible coordination pattern of OHM3 LAt (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 ions also 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 –OPc O2 3 group may be coordinated to the two divalent metal ions (bonded to the phenyl group
O-
AdO
O
O
P Cd
50
P
-O
NH
%
30
N
H4CdLAt
Cd3LAt
20
OHCd3LAt
P NH
-O
40
O
O-
-OOH OM
O -
M
O-
M
H4Cd2LAt N
10
H6L 0 3
4
5
6
7
8
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.)
NH
NH
Fig. 4. The possible binding structure of OHM3 LAt. Coordinated water is not given.
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and the amino group neighboring with benzene), as a result the electrons of the phosphate are strongly withdrawn 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 potentiometric 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 concentrations of metal ions was carried out at I ¼ 0:1 M KNO3 , pH ¼ 8.0 and was followed by 31 P-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 of ATP. The observed first-order rate constants kobs are obtained by linear fit of the plot of logð½ATP=½ATP0 Þ as 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 kobs ½ATP ¼ d½ATP=dt:
ð1Þ
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, OHM3 LAt of 3:1:1 modes and H2 MLAt of 1:1:1 modes are dominant, although in different degrees for different metal ions). That is understandable. Just as what was discussed in Section 3.4, OHM3 LAt is a strong nucleophile (metalbound hydroxide ion) and the phosphate linkage is activated so strongly that the nuleophilic attack can easily take place. As for H2 MLAt, 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 of the hydroxide ion. Furthermore, the phosphate linkage is not so activated as that in OHM3 LAt (in the mode of OHM3 LAt, three metal ions interacted with each other to activate the phosphate linkage; whereas in the mode of H2 MLAt, 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 a much higher kobs than Zn-complex, which may be due to the reason that the relative content of OHCd3 LAt is about three times more than the content of OHZn3 LAt at the experimental conditions. The said reason is understandable 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
Fig. 5. Observation of ATP-hydrolysis by 31 P-NMR spectroscopy as a function of time. Proton-decoupled 31 P-NMR spectra (at 81 MHz) of 3.3 mM ATP, 9.9 mM Cd2þ and 3.3 mM L at an apparent pH of 8.0 in D2 O:H2 O 1:9 at 70 °C recorded at times indicated (in minutes); the chemical shifts are in ppm relative to external 85% H3 PO4 ; the signals are identified by the following symbols: Ta , Tb , Tc for the a-, b-, c-phosphate groups of ATP; Da , Db for ADP; M for AMP; OP for inorganic phosphate. (Ta and Da overlap. The reference of the 31 P signals downfield shifts for about 0.678 ppm.)
R. Ge et al. / Journal of Inorganic Biochemistry 98 (2004) 917–924 Table 4 The first-order rate constants (kobs 104 min1 ) for the hydrolysis of ATP in the presence or absence of L with/without different metal ions M2þ :L:ATP 1:1:1 1:1:1 3:1:1 3:1:1 0:0:1 0:1:1 a
M2þ Zn2 Cd2 Zn2 Cd2 – –
_ AdO O -
O
P
P-
O O
kobs 104 (min1 ) 7.5 8.3 8.5 138 2 0a
70 0.1 °C, I ¼ 0:1 M KNO3 , pH 8.0, [ATP] ¼ 3.3 mM. No hydrolysis was observed during the experimental period of 10 h.
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 that the 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 2 104 to about 0 min1 . This contrasts with the findings of Lehn J.M. that protonated macrocyclic polyamines can catalyze 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 H2 LAt, 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 of OHM3 LAt 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 by steric effect. As a result, ATP hydrolysis has been inhibited by the ligand L. The proposed mechanism of the hydrolysis of ATP by OHM3 LAt is schematically depicted in Fig. 6. First, the phosphate linkage is greatly activated as discussed in Section 3.4. Then, the phosphate is attacked by the hydroxide ion which is in a suitable position for the nucleophilic attack. The positive charges accumulated in the metal-hydroxo cluster stabilize the negativelycharged transition state of ATP hydrolysis (the transition 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 pentacoordinated 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.
_ AdO O
O O
O H HO
M
923
P
O O_
OH
-
O
P
O O
O O
M M
P-
M
P
O O
OH O
M
H HO
M
ADP + HPO42-
Fig. 6. The proposed mechanism of ATP hydroysis by OHM3 LAt. L is not shown.
4. Conclusion One new chelate ligand 2,9-di(60 -a-phenol-n-20 ,50 diazahexyl)-1,10-phenanthroline L was synthesized and fully characterized. The protonation constants of the ligand as well as the supramolecular interactions between 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 31 P-NMR spectra showed that mainly c-P, sometimes b-P, oxygens took part in the coordination with the metal ions (M2þ /ATP/L) or the protonated hydrogen 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 triCd2þ complex and an inhibitory effect on ATP hydrolysis 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 Natural Science Foundation of Tianjin (No. 023605811). References [1] H. Sigel (Ed.), Metal Ions in Biological Systems, Marcel Dekker, New York, vol. 1–11, 1973–1980. [2] L.G. Marzilli, Prog. Inorg. Chem. 23 (1977) 255–378. [3] R.W. Gellert, R. Bau, Met. Ions Biol. Syst. 8 (1979) 1–56. [4] R.B. Martin, Y.H. Mariam, Met. Ions Biol. Syst. 8 (1979) 57–125. [5] N. Str€ater, W.N. Lipscomb, T.K. labunde, B. Krebs, Angew. Chem. Int. Ed. Engl. 35 (1996) 2024–2055. [6] D.E. Wilcox, Chem. Rev. 96 (1996) 2435–2458. [7] N.H. Williams, in: M. Sinnott (Ed.), Comprehensive Biological Catalysis: A Mechanistic Reference, vol. 1, Academic Press, London, 1998, pp. 543–562. [8] A.C. Hengge, in: M. Sinnott (Ed.), Comprehensive Biological Catalysis: A Mechanistic Reference, vol. 1, Academic Press, London, 1998, pp. 517–542. [9] G.J. Narlikar, D. Herschlag, Ann. Rev. Biochem. 66 (1997) 19–41.
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[10] N.H. Williams, J. Am. Chem. Soc. 122 (2000) 12023–12024. [11] M.W. Hosseini, J.-M. Lehn, M.P. Mertes, Hevl. Chim. Acta 66 (1983) 2454–2467. [12] A. Bianchi, C. Giorgi, P. Paoletti, B. Valtancoli, V. Fusi, E. Garcia-Espana, J.M. Llinares, J.A. Ramirez, Inorg. Chem. 35 (1996) 1114–1120. [13] L.F. Lindoy (Ed.), The Chemistry of Macrocyclic Ligand Complex, Cambridge University Press, Cambridge, UK, 1989. [14] M.W. Hosseini, J.M. Lehn, Helv. Chim. Acta 70 (1987) 1312– 1319. [15] A.E. Martell, R.J. Motekaitis, Q. Lu, D.A. Nation, Polyhedron 18 (1999) 3203–3218.
[16] F.P. Tania, W.S. Peter, M.G. Andre, Polyhedron 20 (2001) 2457– 2466. [17] F. Fenniri, M.W. Hosseini, J.M. Lehn, Hevl. Chim. Acta 80 (1997) 786–803. , H. Sigel, Inorg. Chem. 38 [18] S.A.A. Sajadi, B. Song, F. Gregan (1999) 439–448. [19] A.D. Zuberbuhler, T.A. Kaden, Talanta 29 (1982) 201–206. [20] M.W. Hosseini, J.M. Lehn, L. Magiora, B.K. Mertes, M.P. Mertes, J. Am. Chem. Soc. 109 (1987) 537–544. [21] H. Xia, H. Lin, Y. Chen, Chin. J. Inorg. Chem. 10 (1994) 363–369. [22] A.E. Martell, R.M. Smith (Eds.), Critical Stability Constants, vol. 5, Plenum Press, New York, 1975.
Inorganic Biochemistry 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 Shourong Zhu a, Baofeng Ji a, Fenghua Li a, Hongxing Wu a b
a,*
,
a Department of Chemistry, Nankai University, Tianjin 300071, PR China 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 (M ¼ Zn2þ , Cd2þ ) and adenosine-50 -triphosphate (ATP) were studied. The metal complexes of the chelate recognize the nucleotides via multiple interactions similar to those occurring in the center of enzymes. The hydrolysis of ATP was studied with the mononuclear and trinuclear chelate complexes. Ó 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 nucleic 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 metalloenzymatic reactions catalyzed by the ATPases and plays a key role in numerous processes: photosynthesis phosphorylation (chloroplast ATPase), oxidative phosphorylation (mitochondrial ATPase), muscle action (myosin ATPase), etc. However, despite a large knowl*
Corresponding author. Fax: +86-22-23502458 (H. Lin). E-mail address: [email protected] (H. Lin). 1 Present address: Department of Chemistry, The University of Hong Kong, Hong Kong, China. 0162-0134/$ - see front matter Ó 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 in analyzing 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], biomimetic studies [13] and supramolecular catalysis [14]. Successful catalysis by polyamine ligands relies on the recognition and selectivity of the initially bound substrate and the release of the product formed following chemical transformation. In aqueous solution, the stability of the recognition complex is related to hydrogen bonding and coulombic interactions as well as the geometrical ‘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
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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 metal complexes [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 13 P-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.
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 ethylenediaminetetraacetic acid (EDTA) titrations. 2.2. Equipments Elemental analysis was made on a Perkin–Elmer 240C elemental analyzer. The 1 H-NMR, and 31 P-NMR spectra were recorded with a Varian UNITY-plus 400 MHz Spectrometer. IR spectra were obtained as KBr disks on a Euinox 55 FT Spectrometer (Bruker). Electrospray 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. Experimental 2.3. Synthesis of the chelate ligand L (Fig. 1) 2.1. Materials Most of the starting materials were obtained commercially 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 nucleotides were freshly prepared. All other materials used in the experiments including the potassium hydrogen phthalate, HNO3 , KOH, and the metal ion solutions of
2.3.1. Synthesis of 1-a-phenol-n-2,5-diazapentane Ethylenediamine (6 g) was dissolved in ethyl alcohol (20 ml). Salicylaldehyde (1.22 g) dissolved in ethyl alcohol (70 ml) was added dropwise to the above solution during a period of 10 h with stirring at room temperature. 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
N
NH2
NH
CHO
OH
OH
NH2CH2CH2NH2
NH
1) N OHC 2) NaBH4
N
NaBH4
OH
HN
N
HO
N
HO
CHO
H N
NH
NH2
L Fig. 1. The processes of the ligand L preparation.
R. Ge et al. / Journal of Inorganic Biochemistry 98 (2004) 917–924
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 (yield 60.4%). 1 H-NMR (D2 O, ppm): d 6.98, 6.61 (s, 4H, Ph); d 3.92 (s, 4H, CH2 –Ar); d 3.53, 3.40 (s, 8H, N–CH2 –CH2 – N). IR (KBr pellet, cm1 ): 1161 ðmCN Þ; 3423 ðmNH Þ; 456, 865, 1595 (Ar); 2845 (mCH Þ; 3640 ðmOH Þ. Elemental analysis: Calc. for C9 H14 N2 O, H: 8.49%; C: 65.03%; N: 16.85%. Found H: 8.41%; C: 65.12%; N: 16.74%. ESIMS: 166.4 (calculated for C9 H14 N2 O 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-evaporated 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 Na2 SO4 . The mixture was filtered and chloroform 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 ethyl alcohol, recrystallized with ethanol/ether and then dried in a vacuum desiccator. Yield 50.1%. 1 H-NMR (D2 O, 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); d 3.62, 3.50 (s, 8H, N–CH2 –CH2 –N). IR (KBr pellet, cm1 ): 1161 (mCN ); 3423 ðmNH Þ; 456, 865, 1595 (Ar); 2845 ðmCH Þ; 3640 ðmOH Þ; 1586, 1345, 865 (Phen). Elemental analysis: Calc. for C32 H36 N6 O2 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 C32 H36 N6 O2 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 potentiometric titrations Potentiometric determination was measured in a 50 ml jacketed cell thermostated at 25.0 0.1 °C by a refrigerated circulating water bath. Anaerobic conditions were maintained using pre-purified N2 as an inert atmosphere, 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 literature [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
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Kx ¼ 1:008 1014 , cþ H ¼ 0:825 of water were used for the calculation. The calculations were carried out by TITFIT, a Newton–Gauss–Marquardt nonlinear leastsquares program [19]. The final results are the averages of three independent titrations, each titration containing about 50 experimental points. 2.5. Kinetics Kinetic studies were performed by following the time evolution of the 31 P-NMR spectra. Since 31 P-NMR signals of ATP, ADP, AMP and orthophosphate (OP) are distinct, ATP-hydrolysis can be monitored conveniently and accurately by following the changes in the concentrations of various species [20]. Eight-five percent H3 PO4 was used as an external standard. In a typical experiment, a 0.6 ml solution (10% D2 O/H2 O) in a 5 mm tube containing 3.3 mM ATP and 3.3 mM L and/or corresponding concentrations of metal ions was adjusted 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 in Fig. 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 protonated in the pH range studied (2–10.5), due to the lower pK H H-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 density of phen. The six protonation constants listed for the ligand 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) and ortho-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 pbenzene, the amino groups neighboring with phen have Table 1 The protonation constants for the chelate ligand L Equilibria
L
[LH]/[L][H] [LH2 ]/[LH][H] [LH3 ]/[LH2 ][H] [LH4 ]/[LH3 ][H] [LH5 ]/[LH4 ][H] [LH6 ]/[LH5 ][H]
10.19 0.05 9.75 0.03 8.88 0.05 7.54 0.06 5.50 0.03 4.66 0.02
25.0 0.1 °C, I ¼ 0:1 M KNO3 , [L] ¼ 0.5 mM.
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R. Ge et al. / Journal of Inorganic Biochemistry 98 (2004) 917–924 100 90
H4L
H6L
80
H3L
70
H5L
60
H2L
%
50 40
HL
30 20
L
10 0 2
4
6
8
10
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.)
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 other two constants should belong to the amino groups neighboring with phen. Owing to the build-up of positive charge on the chelate ligand and the consequentially increased coulombic repulsion for an additional proton, each successive protonation becomes weaker, as reflected 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, pyrophosphate 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 potentiometric equilibrium methods and the binding constants 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 repTable 2 The stability constants of the ligand with respective ATP, ADP or AMP Equlibria
ATP
ADP
AMP
LgbH6 NuL LgbH5 NuL LgbH4 NuL LgbH3 NuL LgbH2 NuL LgbHNuL
53.71 48.96 43.96 38.57 32.21 25.76
52.45 47.85 43.02 37.62 31.57 25.11
46.65 42.13 36.71 30.68 24.79
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, since the 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 nucleotides by the chelate ligand via multiple interactions relies on the length of phosphate chain and the charge of substrates. 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 catalytic conversion 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 :L ¼ 3:1 as an example) may be as follows: In the low pH range, the mode H4 ML 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 Table 3 The binary stability constants of the ligand L with respective M2þ (Zn2þ or Cd2þ ) in the absence or presence of ATP M2þ :L:At (a) 1:1:0
3:1:0
(b) 1:1:1
3:1:1
Equlibria
Zn2þ
Cd2þ
LgbH4 ML LgbH3 ML LgbH2 ML
41.53 32.57 23.05
40.58 31.29 17.85
LgbH4 ML LgbH2 M2 L LgbM3 L LgbOHM3 L
41.53 31.64 20.81 13.77
40.58 30.39 19.89 13.65
LgbH5 MLAt LgbH4 MLAt LgbH3 MLAt LgbH2 MLAt
57.09 50.98 46.12 42.19
55.19 49.42 45.36 37.51
LgbH4 MLAt LgbH2 M2 Lat LgbM3 LAt LgbOHM3 LAt
57.34 54.77 47.89 41.97
56.13 52.61 46.94 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
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 H2 M2 L. With the further increase of pH, all the protonated protons are lost and the third metal ion takes part in the coordination and forms another chelate ring. When pH increases further, one of the water coordinated with the metal ion deprotonates to give the monohydroxyl complex OHM3 L 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 XII group 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 H4 CdLAt, H2 Cd2 LAt, Cd3 LAt and OHCd3 LAt (At represents ATP). Complex Cd3 LAt is dominant at around pH 5.5, whereas OHCd3 LAt is a major species at pH > 7.5. Comparing the binding constants of the M2þ /ATP/L with the corresponding constants of M2þ /L, considerable interactions apparently took place when the nucleotide was introduced to the 1:1 and 3:1 (M2þ :L)
60
921
systems. The nucleotides can offer additional coordination sites to metal ion and thus increase the stability of the ternary complexes. We measured the 31 P-NMR spectra of ATP, 1:1 (L:ATP) as well as 3:1:1 (Cd2þ :L:ATP) systems at pH 8.0. 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, compared with the chemical shifts of ATP alone. At this experimental condition, HLAt is dominant in the system with a little amount of H2 LAt. The chemical shift upon the binding of ATP by protonated L is induced mainly by the electrostatic interactions and the formation of hydrogen 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 protonated phenyl group in HLAt. b-P may also take part in the interaction in H2 LAt. As to 3:1:1 (Cd2þ :L:ATP) system studied, OHM3 LAt is dominant in the system and ATP’s three signals shift 0.295, 2.156 and 5.284 ppm, 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 possible coordination pattern of OHM3 LAt (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 ions also 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 –OPc O2 3 group may be coordinated to the two divalent metal ions (bonded to the phenyl group
O-
AdO
O
O
P Cd
50
P
-O
NH
%
30
N
H4CdLAt
Cd3LAt
20
OHCd3LAt
P NH
-O
40
O
O-
-OOH OM
O -
M
O-
M
H4Cd2LAt N
10
H6L 0 3
4
5
6
7
8
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.)
NH
NH
Fig. 4. The possible binding structure of OHM3 LAt. Coordinated water is not given.
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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 withdrawn 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 potentiometric 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 concentrations of metal ions was carried out at I ¼ 0:1 M KNO3 , pH ¼ 8.0 and was followed by 31 P-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 of ATP. The observed first-order rate constants kobs are obtained by linear fit of the plot of logð½ATP=½ATP0 Þ as 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 kobs ½ATP ¼ d½ATP=dt:
ð1Þ
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, OHM3 LAt of 3:1:1 modes and H2 MLAt of 1:1:1 modes are dominant, although in different degrees for different metal ions). That is understandable. Just as what was discussed in Section 3.4, OHM3 LAt is a strong nucleophile (metalbound hydroxide ion) and the phosphate linkage is activated so strongly that the nuleophilic attack can easily take place. As for H2 MLAt, 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 of the hydroxide ion. Furthermore, the phosphate linkage is not so activated as that in OHM3 LAt (in the mode of OHM3 LAt, three metal ions interacted with each other to activate the phosphate linkage; whereas in the mode of H2 MLAt, 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 a much higher kobs than Zn-complex, which may be due to the reason that the relative content of OHCd3 LAt is about three times more than the content of OHZn3 LAt at the experimental conditions. The said reason is understandable 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
Fig. 5. Observation of ATP-hydrolysis by 31 P-NMR spectroscopy as a function of time. Proton-decoupled 31 P-NMR spectra (at 81 MHz) of 3.3 mM ATP, 9.9 mM Cd2þ and 3.3 mM L at an apparent pH of 8.0 in D2 O:H2 O 1:9 at 70 °C recorded at times indicated (in minutes); the chemical shifts are in ppm relative to external 85% H3 PO4 ; the signals are identified by the following symbols: Ta , Tb , Tc for the a-, b-, c-phosphate groups of ATP; Da , Db for ADP; M for AMP; OP for inorganic phosphate. (Ta and Da overlap. The reference of the 31 P signals downfield shifts for about 0.678 ppm.)
R. Ge et al. / Journal of Inorganic Biochemistry 98 (2004) 917–924 Table 4 The first-order rate constants (kobs 104 min1 ) for the hydrolysis of ATP in the presence or absence of L with/without different metal ions M2þ :L:ATP 1:1:1 1:1:1 3:1:1 3:1:1 0:0:1 0:1:1 a
M2þ Zn2 Cd2 Zn2 Cd2 – –
_ AdO O -
O
P
P-
O O
kobs 104 (min1 ) 7.5 8.3 8.5 138 2 0a
70 0.1 °C, I ¼ 0:1 M KNO3 , pH 8.0, [ATP] ¼ 3.3 mM. No hydrolysis was observed during the experimental period of 10 h.
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 that the 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 2 104 to about 0 min1 . This contrasts with the findings of Lehn J.M. that protonated macrocyclic polyamines can catalyze 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 H2 LAt, 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 of OHM3 LAt 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 by steric effect. As a result, ATP hydrolysis has been inhibited by the ligand L. The proposed mechanism of the hydrolysis of ATP by OHM3 LAt is schematically depicted in Fig. 6. First, the phosphate linkage is greatly activated as discussed in Section 3.4. Then, the phosphate is attacked by the hydroxide ion which is in a suitable position for the nucleophilic attack. The positive charges accumulated in the metal-hydroxo cluster stabilize the negativelycharged transition state of ATP hydrolysis (the transition 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 pentacoordinated 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.
_ AdO O
O O
O H HO
M
923
P
O O_
OH
-
O
P
O O
O O
M M
P-
M
P
O O
OH O
M
H HO
M
ADP + HPO42-
Fig. 6. The proposed mechanism of ATP hydroysis by OHM3 LAt. L is not shown.
4. Conclusion One new chelate ligand 2,9-di(60 -a-phenol-n-20 ,50 diazahexyl)-1,10-phenanthroline L was synthesized and fully characterized. The protonation constants of the ligand as well as the supramolecular interactions between 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 31 P-NMR spectra showed that mainly c-P, sometimes b-P, oxygens took part in the coordination with the metal ions (M2þ /ATP/L) or the protonated hydrogen 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 triCd2þ complex and an inhibitory effect on ATP hydrolysis 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.
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