Nucleic Acids Research 2 February 1977 Volume 44 Number ubr2Fbur 97NcecAisRsac
Voum
A 31 p - NMR study of the interaction of Mg2+ ions with nucleoside diphosphates
S.Tran-Dinh and J.M.Neumann Departement de Biologie - Centre d'Etudes Nucleaires de Saclay - BP N02 - 91190 Gif-sur-Yvette, France Received 29 November 1976
ABSTRACT : The interaction of Mg2 with nucleoside disphosphates . ADP, GDP, CDP and UDP has been studied by phosphorus magnetic resonance spectroscopy in aqueous solution. The results show that these four nucleotides behave similarly, the Mg2+ ion binds to the a but not to the 0 phosphate moiety. The strength of the interaction of Mg2+ ions with nucleoside diphosphates is weaker than with nucleoside triphosphates. The association of Mg2+ on the phosphate chain is stronger in a neutral than in an acid medium. INTRODUCTION : The nucleoside di- and triphosphates play an important role in the process of energy transfer in living organisms. Generally they participate as substrates in numerous enzymatic reactions in vivo and in vitro. Without doubt, divalent ions, in particular Mg2+, exert as activators, a large and often determining influence on the kinetics of these reactions. Indeed, in the last fifteen years, the interaction between divalent ions and nucleoside di- and triphosphates, in particular the adenosine di- (ADP) and tri- (ATP) phosphates (1-9), has been studied extensively. COHN and HUGUES (1) concluded from a 31P - and 1H - NMR study that in aqueous solution, Mn2 , Co2 , Ni2+, bind to all three phosphate groups Pa, Pa, Pr of ATP, Mg 2, Ca 2, Zn2+ to P and P only. Concerning ADP, Mg 2, Mn2, Cu2+ interact, according to these authors, with both the a and a phosphate groups. However, TRAN-DINH et al (10) showed in a recent 31P-NMR study that at both neutral and acid pH, Mg2+ binds not to the y but to the a phosphate group of ATP, GTP, CTP and UTP. The chemical shift variation of P , observed by COHN and HUGUES in the absence and presence of Mg ions, is simply due to the pK variation of this group (Py) by about 1.5 units (10). It is C Information Retrieval Umited 1 Falconberg Court London W1V 5FG England
397
Nucleic Acids Research of interest to reinvestigate the interaction of the Mg2+ ion on the phosphate chain of nucleoside diphosphates while taking into consideration a possible pK change of the P when adding divallent ions. In this paper, we should like to report a quantitative phosphorus magnetic resonance study of Mg2+ complexing with four nucleoside diphosphates ADP, GDP, CDP and UDP. The results the Mg2+ binyield information on the nature of the complex ding site and the conformation of the phosphate chain in the ions. presence of
now
Mg2
MATERIALS AND METHODS The nucleoside diphosphates ADP, GDP, CDP, UDP of highest grade were purchased from P.L. Laboratories and magnesium chloride (reagent grade MgCl2.6H20 crystals) from MERCK. Divalent ion impurities were systematically removed by shaking D20 solutions of nucleotide with chelex 100. The pH was adjusted with concentrated solutions of DCl or NaOD and measured with a Tacussel pH meter. The pD was taken to be equal to pH + 04 (11). The 31P-NMR spectra were recorded at 40.5 MHz on a Varian XL100-12WG spectrometer at about 300C. Proton noise decoupling was obtained with a varian gyrocode spin decoupler. An acquisition time of 3 sec was used throughout this work (resolution = 0.3 Hz). For all the measurements, D20 used as solvent provided the deuterium lock signal. The 31p chemical shifts of nucleotides (in ppm) are measured from a 85% H 3PO4 external reference. RESULTS AND DISCUSSION : The chemical shifts of phosphorus and 31 p - 31 p coupling constant Ja Sof the four nucleotides ADP, GDP, CDP, UDP, were concentration at constant determined as a function of the Mg nucleoside diphosphate concentration (0.05M) and constant pD between 8.2 and 9.2. In general, when the ratio R = [Mg2 ] [nucleotide] varies from 0 to 1, the chemical shift variation observed is comparatively large for Pa and small for P . When R = 1 -* 2, virtually no change in chemical shift or coupling 2+ constant J , was observed. In the absence of Mg , the four 2
nucleotides studied behave similarly. In the presence of Mg GDP is less soluble than its analogues but the chemical shifts
398
Nucleic Acids Research and coupling constant J.-, are not very different. Fig. 1 shows the phosphorus spectra (with proton noise decoupling) of ADP in the absence and presence of a stoichiometric quantity for Mg2+ ions at neutral pH. When R varies from 0 to 1, P of the four nucleotides studied is shifted towards lowfields 0.2 Hz to by 0.5 - 0.7 ppm, while Ja 6 decreases from 22.2 17.3 0.5 Hz. If the Ja f values observed are plotted against 2+ P chemical shift, with different value of R = [MgJ ]/[nuclemeans that a straight line is obtained (Fig.2). This otide], under such conditions the variations of 6 and of Ja f are concomitant and result, in fact from Mg2+ binding on the phosphate chain. ±
±
,
I
I
I
I
I
a
1/
[ADP]
=
[Mg2,] = D
0.05 M 0.05 M 8.6
p0
[ADP]
=
pD
=
0.05 M 9.0
S~~~~~ A.
l~~~~~~~~~~~~~I-.
5
Figure 1:
31P
-
6
7
8
9
NMR spectra of ADP (0.05M.pD
and presence of Mg
ions
10
-
11
6(ppm)
8.6,30°C) in the absence
(0.05M). 399
Nucleic Acids Research aa(ppm)
I
ADP
10.5
(0.05M,
pD-8.4)
[ADP]
10.4 10.3
-
-
10.2_ 10.1 R=1
10.0
18.0
19.0
20.0
21.0
22.0
Ja-13(Hz) Figure 2
Chemical shift of
Pa
Ja
versus
The four nucleoside diphosphates were also titrated in the absence and presence of Mg 2+ Fig. 3 shows the typical titration curves of ADP and Table I sums up the phosphorus che31P coupling constants of the four numical shifts and 31P cleoside diphosphates investigated. In the absence and presence of Mg2+ ions, the 6 variation is found to be comparable before .
-
(pD
8.5)
and
after
(pD
4)
protonation
of
the
6 phosphate
only the pK values differing by 1.4 units as in the case of nucleoside triphosphates (10). This explains why COHN and HUGUES (1) observed a difference of 2.7 and 2.3 ppm for PS at pH = 5.3 et 6.8 (pD = 5.7 and 7.2) respectively. The variation of Ja 6 as a function of pD for the typical case of CDP in the absence and presence of Mg2+ ions is shown in Fig.4. It is interesting to note that when the a phosphate 2 decreases by group is protonated, in the absence of Mg2, a increases by aabout 2Hz, while in the presence of Mg 2, J bout 1 Hz. Similar results have been obtained by ELLENBERGER 2+ et al (12) for ADP in the absence of Mg . Moreover it may be group,
seen
400
in
table
I
that
in
an
acid
medium
(pH
4)
the
respective
4,
6(ppm) 13
Nucleic Acids Research
AOP ( 0.OSM)
12 11 10
9
8
7
Mg2 (o.5 M)
-ADP
.......ADP.Mg2 1,
6
5~
2
Figure 3
31
3 P titration
curves
a
6
5
4
3
9
0
11
pD
of ADP in the absence and presence of Mg
2+
TABLE I
31P
chemical shifts (in ppm upfield from 85% H3PO4 as externel standard) and 31P-31P coupling constants (in Hz) of nucleoside diphosphates in the absence and presence of Mg2 (R - cMg2 / [Nucleotide)) at neutral and acid pH. NEUTRAL pH
ACID pH I
RpD ADP
(0.05
1i)
6,
6-
JQBpD
6
6B
JC8
0 9.2 10.45 5.94 22.2 4.3 11.06 10.54 20.2 1 8.2 9.960 5.80 17.5 3.9 11.17 10.45 18.3
2+ -2-
A6, AJ GDP
0.49 0 9.5 10.51 9.97
(0.05 p) 1 9.0
0.14
4.7
-0.11
0.09
1.9
5.96 22.3 4.1 11.11 10.55 20.3 6.13 16.8 3.4 11.32 10.81 17.5
2+ -2-
A6, AJ
0.54 -0.17
0 9.2 10.63 CDP (0.05 ii) 1 8.6 9.92
A6, AJ UDP
0.71 0 9.2 10.64 9.97
(0.05 p) 1 8.8
A6, AJ
0.67
5.5
0.21
0.26
2.8
6.01 22.5 4.0 11.19 10.62 20.1 5.80 17.7 3.6 11.26 10.70 18.7
0.21
4.8
-0.07 -0.08
1.4
6.00 22.4 3.0 11.14 10.62 20.2 5.79 17.9 3.1 11.29 10.81 18.6 0.21
4.5
-0.15 -0.19
1.6
401
Nucleic Acids Research values of
P.,
chemical shifts and the Ja f coupling constant are very similar in the absence and presence of Mg ions. From these different results we conclude that : - At neutral pH, the Mg 2+ ion binds not to the a but only to the a phosphate group. The interaction arises from 1 : 1 metal nucleotide complexing. - After protonation of the a phosphate group (pH=4) the P
association of the Mg2+ ion on the phosphate chain, if it does exist, is very weak. In addition the fact that the variation of 6 is much larger than that of 6 suggests that the Mg2 ion binds not to the two oxygen atoms of the phosphate chain (R - 0 - Pa - 0 P) but to the other two oxygen atoms of the a phosphate group, otherwise the P chemical shift variation, which is very sensitive to environment, should be equal at least to half A6a Finally some remarks are called for : 1) Although the terminal phosphate group of nucleoside di and tri-phosphates has two negative charges at neutral pH, it is surprising to note that the Mg2 ion binds not to this group but to the preceding one (P for nucleoside triphosphates -
Ja-3(Hz) 23 L ' 22-
21-
20
.
.
CDP
* CDP+Mg2+ 19A
-0 Figure 4
pH dependence of the
P
-
pD
P coupling constant J
2+. O of Mg in the absence and (0.05M) presence
402
ions
a
of CDP
(0.05M).
Nucleic Acids Research and
Pa
for nucleoside diphosphates). 2) In the absence and presence of
riation
of
nucleoside
with that of (AJ
4.0
Ja
Hz)
diphosphates
6 and J
while
(A
Mg2
J
4.7
ions the J Hz)
is
va-
comparable
y for nucleoside triphosphates (10) (=
A6a
0,5
0.7
ppm)
of
the
former
catego-
ry of compounds is three or four times smaller than A6a(=2.2-2.3 ppm) of the latter. Thus it seems likely that in the presence of ions, the conformation of the phosphate chain (R - 0 - PO - P) is similar for both families of compounds while the polarisation for the binding site (Pa for diphosphates and P for triphosphates) is quite different. This suggests that the strength of the interaction of Mg2 ions with nucleoside diphosphates is weaker than with nucleoside triphosphates. On may imagine that in the case of the nucleoside diphosphates, one water molecule bridges the a phosphate moiety and the Mg2 ion.
Mg2
REFERENCES. 1) COHN, M. and HUGUES, T.R. (1962) J. Biol. chem. 237, 176181. 2) STERNLICHT, H., JONES, D.E. and KUSTIN, K. (1968) J. Amer. Chem. Soc. 90, 7110 - 7118. 3) GLASSMAN, T.A., COOPER, C., HARRISON, L.W., and SWIFT, T.J., (1971) Biochemistry 10, 843-851. 4) KUNTZ, G.P.P., GLASSMIN, T.A., COOPER, C., and SWIFT, T.J., (1972) Biochemistry 11, 538-541. 5) LAM, Y.F., KUNTZ, G.TF'P., and KOTOWYCZ, G., (1974) J. Amer. Chem. Soc. 96, 1834 - 1839. 6) HEYDE, M.E., and RIMAI, L., (1971) Biochemistry 10, 1121 1128. 7) SIGEL, H., (1975) J. Amer. Chem. Soc. 97, 3209 - 3214. 8) BANYASZ, J.L., and STUEHR, J.E., (19737-J. Amer. Chem. Soc. 95, 7226-7231. 9) TTNSWELL, P., THORNTON, J.M., KORDA, A.V., and WILLIAMS, R.J.P., (1975) Eur. J. Biochem. 57, 135 - 145. 10) TRAN-DINH, S., ROUX, M. and ELLENBERGER, M., (1975) Nucleic Acids
Res.
2,
1101
-
1110.
11) GLASOE, P.K. and LONG, F.A., (1960) J. Phys. Chem. 64, 188 192. 12) ELLENBERGER, M., BREHAMET, L., VILLEMIN, M. and TOMA, F., (1970) F.E.B.S. Letters 8, 125 - 128.
403