to all three phosphate groups P, P Q, P of ATP, Mg +, Ca +, p

Transcription

1 Volume 4 Number 2 February 1977 Nucleic Acids Research A 31 P - NMR study of the interaction of M^2" 1 " ions with nucleoside diphosphates S.Tran-Dinh and J.M.Neumann Departement de Biologie - Centre d'etudes Nucleaires de Saclay - BP N Gif-sur-Yvette, France Received 29 November 1976 ABSTRACT : The interaction of Mg 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 B 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 Mg^+, 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 31 P - and 1 H - NMR study that in aqueous solution, Mn +, Co +, Ni, bind to all three phosphate groups P, P Q, P of ATP, Mg +, Ca +, p?+ ' 2+?+ 7+ Zn to Pg and P only. Concerning ADP, Mg, Mn, Cu interact, according to these authors, with both the a and g phosphate groups. However, TRAN-DINH et a_l (10) showed in a recent 31 P-NMR study that at both neutral and acid ph, Mg binds not to the Y but to the 3 phosphate group of ATP, GTP, CTP and UTP. The chemical shift variation of P, observed by COHN and HUGUES 2 + 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 Information Retrieval Limited 1 Falconberg Court London W1V5FG England

2 now of interest to reinvestigate the interaction of the Mg 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 Mg complexing with four nucleoside diphosphates ADP, GDP, CDP and UDP. The results yield information on the nature of the complex, the Mg binding site and the conformation of the phosphate chain in the presence of Mg + ions. MATERIALS AND METHODS : The nucleoside diphosphates ADP, GDP, CDP, UDP of highest grade were purchased from P.L. Laboratories and magnesium chloride (reagent grade MgCl 2.6H 2 O crystals) from MERCK. Divalent ion impurities were systematically removed by shaking D 2 0 solutions of nucleotide with chelex 100. The ph was adjusted with concentrated solutions of DC1 or NaOD and measured with a Tacussel ph meter. The pd was taken to be equal to ph + 04 (11). The 31 P-NMR spectra were recorded at 40.5 MHz on a Varian XL100-12WG spectrometer at about 30 c C. Proton noise decoupling was obtained with a varian gyrocode spin decoupler. An acquisition time of 3 sec was used throughout this work (resolution a 0.3 Hz). For all the measurements, D 2 O used as solvent provided the deuterium lock signal. The 31 p chemical shifts of nucleotides (in ppm) are measured from a 854 H PO RESULTS AND DISCUSSION : constant J external reference. The chemical shifts of phosphorus and p - p coupling Q of the four nucleotides ADP, GDP, CDP, UDP, were P 2 + determined as a function of the Mg concentration at constant nucleoside diphosphate concentration (0.05M) and constant pd between 8.2 and 9.2. In general, when the ratio R = ^Mg + J / [nucleotide] varies from 0 to 1, the chemical shift variation observed is comparatively large for P and small for Pg. When R = 1-2, virtually no change in chemical shift or coupling constant J o was observed. In the absence of Mg, the four a ~p 2 + nucleotides studied behave similarly. In the presence of Mg, GDP is less soluble than its analogues but the chemical shifts 388

3 and coupling constant J a _ 6 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 Mg ions at neutral ph. When R varies from 0 to 1, P of the four nucleotides studied is shifted towards lowfields by ppm, while J a _ g decreases from 22.2 ± 0.2 Hz to 17.3 ± 0.5 Hz. If the J o values observed are plotted against P a chemical shift, with different value of R = [Mg j/[nucleotide], a straight line is obtained (Fig.2). This means that under such conditions the variations of 6 and of J are con- 2+ a a " B comitant and result, in fact, from Mg binding on the phosphate chain. [ADP] = 0.05 M [Mg 2 '] = 0.05 M pd = 8.6 [ADP] «0.05 M pd > ( ppm) Figure 1 : 'P - NMR spectra of ADP (0.05M.pD - 8.6,30*C) in the absence and presence of Mg ions (0.05M). 399

4 5a(ppm) Ji 11/ 1 i i _ ADP (0.05M, pd = 8' R= [Mg *J [ADP] / R= / / Figure 2 : Chemical shift of P versus J a a a-b i i Ja-/3(Hz) The four nucleoside diphosphates were also titrated in the absence and presence of Mg +. Fig. 3 shows the typical titration curves of ADP and Table I sums up the phosphorus chemical shifts and 31 P - 31 P coupling constants of the four nucleoside diphosphates investigated. In the absence and presence of Mg ions, the 6 g variation is found to be comparable before (pd =8.5) and after (pd = 4) protonation of the 6 phosphate group, 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 P g at ph = 5.3 et 6.8 (pd = 5.7 and 7.2) respectively. The variation of J o as a function of pd for the typical a-p 2 + case of CDP in the absence and presence of Mg ions is shown group in Fig.4. is protonated, It is interesting in the absence to note of that Mg when the B phosphate about 2Hz, while in the presence of Mg -B decreases by ob i ncreases by a- bout 1 Hz. Similar results have been obtained et al (12) for ADP in the absence of Mg +. Moreover by ELLENBERGER it may be seen in table I that in an acid medium (ph = 4) the respective 400

5 filppm) 13. ADPI0.05M) -go P.. w J i 4 1 K n pd Figure 3 : P titration curvea of ADP in the absence and presence of Mg TABLE I 31 P chenical shifts (in ppn upfield from 851 H,PO. as external T 1 T standard) and -"p--' p coupling constants (in Hz) of nucleoside diphosphates in the absence and presence of Mg (R - CMg + D / CNucleotide]) at neutral and acid ph. NEUTRAL ph ACID ph R pd J a 4 B VB pd S a 4 B V 6 ADP (0.05 i) *Mg2* AS, AJ GDP (0.05 u) * Mg 2 ' A4, AJ SS S1C CDP (0.05 u) * Mg 2 * A«, AJ S UDP (0.05 p) * Mg 2 * 46, AJ

6 values of P Q, P g chemical shifts and the J a _ 6 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 + ion binds not to the 6 but only to the a phosphate group. The interaction arises from 1 : 1 metal nucleotide complexing. - After protonation of the 6 phosphate group (ph=4) the association of the Mg 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 Mg ion binds not to the two oxygen atoms of the phosphate chain (R-O-P Pg) 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 A6. - 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 Mg ion binds not to this group but to the preceding one (P g for nucleoside triphosphates Jo-/3(Hz) Figure 4 : ph dependence of the P - P coupling constant J _ of CDP (0.05M) in the absence and presence of Mg ions (0.05M). 402

7 and P a for nucleoside diphosphates). 2) In the absence and presence of Mg ions the J _ variation of nucleoside diphosphates (A J = 4.7 Hz) is comparable with that of J _ and J g for nucleoside triphosphates (10) (AJ =4.0 Hz) while A& a (= 0,5-0.7 ppm) of the former category of compounds is three or four times smaller than A6 g (= ppm) of the latter. Thus it seems likely that in the presence of Mg ions, the conformation of the phosphate chain (R P Pg) is similar for both families of compounds while the polarisation for the binding site (P for diphosphates and P g for triphosphates) is quite different. This suggests that the strength of the interaction of Mg ions with nucleoside diphosphates is 'weaker than with nucleoside triphosphates. On may imagine that in the case of the nucleoside diphosphates, one water molecule br: bridges the a phosphate moiety and the Mg ion. REFERENCES. 1) COHN, M. and HUGUES, T.R. (1962) J. Biol. chem. 237, ) STERNLICHT, H., JONES, D.E. and KUSTIN, K. (1968) J. Araer. Chem. Soc. 90, ) GLASSMAN, T.A., COOPER, C, HARRISON, L.W., and SWIFT, T.J., (1971) Biochemistry 10, ) KUNTZ, G.P.P., GLASSRSN, T.A., COOPER, C, and SWIFT, T.J., (1972) Biochemistry 11, ) LAM, Y.F., KUNTZ, G.PTP., and KOTOWYCZ, G., (1974) J. Amer. Chem. Soc. 6, ) HEYDE, M.E., and RIMAI, L., (1971) Biochemistry 10, ) SIGEL, H., (1975) J. Amer. Chem. Soc. 97, ) BANYASZ, J.L., and STUEHR, J.E., (1973"T~J. Amer. Chem. Soc. 95, ) TA~NSWELL, P., THORNTON, J.M., KORDA, A.V., and WILLIAMS, R.J.P., (1975) Eur. J. Biochem. 57, ) TRAN-DINH, S., ROUX, M. and ELLENlERGER, M., (1975) Nucleic Acids Res. 2, ) GLASOE, P.K. and LONG, F.A., (1960) J. Phys. Chem. 64, ) ELLENBERGER, M., BREHAMET, L., VILLEMIN, M. and TOMA, F., (1970) F.E.B.S. Letters 8,

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