TSINGHUA SCIENCE AND TECHNOLOGY ssN100702142325pp43-445 Volume 5, Number 4, December 2000 Interaction of Divalent Metal lons with the Adenosine Triphosphate Measured using nuclear magnetic resonance LUO Xuechun(罗雪春),zHAO№ anming(赵南明), ZHANG Riqing(张日清) State Key Laboratory of Biomembrane and Membrane Biotechnology Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China Abstract The interaction of adenosine triphosphate with divalent metal ions is important in biochemical functions. The effects of pH and metal ions Mg +, Ca?+, Zm+, Mn?+, and Co?+ on the chemical shift of the phosphate group of ATP have been studied using Nuclear Magnetic Resonance. The chemical shift of the B-phosphate of ATP is the most sensitive to pH. Ca and Mg bind with the a- and B-phosphate groups of ATP Zn+ binds to the adenosine ring hydrogen as well as to phosphate. The paramagnetic ions Mn+ and Co* do not cause chemical shifts of the phosphate or proton peak. Mn+ and Co*t broaden the resonance peak only Key words ATP: divalent metal ion: Nuclear Magnetic Resonance( NMR The interaction of adenosine triphosphate with diva-water lent metal ions is important in biochemical functions. The intracellular free Mg2+ can be estimated- by separating 1.2 NMR measurement the a- and B-phosphate peaks of the ATP NMRP spec- NMR spectra were measured on a Bruker AM 500 trum and by knowing the MgATP dissociation constant. For the 3P measurement, Fo=202.459 MHz, spectrum The need for divalent metal ions for all enzymatic reac- width was 5400 Hz, 90 degree pulse width was 15 ms tions involving ATP is well known, but the role of the with 100 scans for 0. 1 mol atp solution and 10 000 metal ions is not cleara scans for 0. 01 mol solution The chemical shift of the P This study was conducted to obtain infomation con- peak in 85%H3 PO4 was selected as zero as a chemical ceming changes in the structure of adenosine triphosphate shift reference caused by interaction with divalent metal ions Mg? Figure 1 shows a high resolution spectrum Ca2+, zn2+, Mn2+, and Co2+.The information on the 0. 1 mol ATP solution at p The two peaks at low structural changes is obtained from the field are due to the y-phosphate group of ATP. The split chemical shift and the line width ofP and H spectra peak is caused by spin-spin coupling interaction with B- 1 Materials and Method phosphate. The spin-spin coupling coefficient=19 Hz. The ATP B-phosphate resonance peak is split into 1.1 Sample preparation phosphate. The a-phosphate peak was split by the B- The crystalline disodium salt of ATP was purchased phosphate as well as H, on Cn of ribose from Sigma. ATP solution (0. 1 mol and 0. 01 mol in For the H measurement, Fo 500. 134 MHz %D20+90%H2O)was adjusted to pH 7.0.0.6 mL spectrum width was 5 154 Hz, pulse width was 5 ms with of the solution was transferred into a 5 mm diameter NMR 16 scans. The Homo-Nuclear Presaturation program was tube to the 3p and h nmr igal. the che cal shift of The ing solution for the divalent metal ions was the water peak is 4. x 10- as a chemical shift reference prepared by dissolving analytical grade metal chloride in the H Nmr measurement Received: 1999-04-26: revised: 2000-04-25 Figure 2 shows a H spectrum at low field. From low to high field, the peaks represent Hs and H2 on Cg, Con
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444 Tsinghua Science and Technology, December 2000, 5(4):443-445 adenosine, and H on CI of ribose Table 2 Phosphate chemical shift(8)of ATP for various Ca+ concentrations(ESD =+0.03) [Ca2+](mV/L) 3.153.258.298.3818.518.6318 1.03.143.238.288.3718.5118.6018.69 3.133.228.278.3718.4718.5618.65 3.153.248.298.3818.4918.5818.67 143.238.298 8.3318.4218.51 963.058.228.3017.5417.6317.71 Fig 1 P spectrum for 0.1 mol/L ATP solution (crystalline disodium salt of ATP in 10% D,0+90% H,0) and zn2+ had the same results, These results and Zn+ mostly bind to the a- of atp with a few binding to th group. Figure 3 shows the effect of Cat on theP spec From low to high field the peaks are Hg on Cg 2 Results 2.1 Effect of ph on chemical shift The chemical shift of the phosphate peak different pH values, Table 1 Fig 3 3P spectra of ATP solution with 5x 10-3mol/L Table 1 Phosphate chemical shift(8) at different pH Ca+ doping( top )and without Ca?+ doping(bottom (EsD.=±0.03 2. 3 Interaction of Mg+, Ca2+, and zn+ with ATP hydrogen 6.174.945.078.448.5319.1119.2119.30 6.334.684.78.448.5419.0819.1719.27 able 3 lists the chemical shift of proton Hg 6.623.893.998.368.4518.7818.818.97 3.558.358.4418.69187918.85 and H, of ATP for different Ca doping concentra 253.358.368.4218 The results show that the ca+ concentration had Table 1 shows that the chemical shift of the B-phos phate is the most sensitive to ph change. In the H spec- Table 3 Chemical shift(8) of H, H, and H, for tra the chemical shift of Hg, H2, and H, did not change various Ca?+ concentrations(Es.p=+0.03) as the ph change [Ca2·](ml/L) Considering the effect of pH on chemical shift of 5.91 5 phosphate, the ph was controlled at 7. 00 throughout the 8.31 following studi 2.7 8.32 7.98 2.2 Binding of Mg t, Ca+and Zn with phos 5.0 8.30 7.95 5.91 phat of ATP 10. 8.30 Table 2 lists the phosphate chemical shift of ATP in 0 I mol ATP solution for various doping Ca+concentra- 5.89
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LUO Xuechun(罗雪春)etal: Interaction of Divalent Metal Ions with the Adenosine…… 445 M?+ produced the same result as Ca2+, but Zn2 caused a chemical shift of the adenine ring Hs as shown in Table 4 and Fig 4 Table 4 Chemical shift(8) of H, H, and H,for various Zn+concentrations(Es.D =+0.03) [zr2·](mml/L) HI 5.92 8.33 10.0 35.0 5.92 5.91 67.0 100.0 8.48 5.91 167.0 8.48 5 5.96 Fig 5 P spectra of ATP solution with 5 x 10-moV/L do doping( bott Fig4 H spectra of ATP solution with 5 x 10-3mol/L Fig.6 'H spectra of ATP solution with 5x 10-5 mol/L ZI+doping( top ) and without Zn?'+ doping(bottom Mn+ doping( top)and without M?++ doping(bottom) Zn caused a chemical shift of the adenine ring Hg (2)Mg2+, Ca2+, and Zn+ form complexes with 0.18x 10-6 lower in the field due to zn binding to the a-and B-phosphate groups of adenosine triphosphate e adenine ring of ATP (3)Zn2+ binds to the adenine ring as well as th atP phosphate group which can explain the physiologic 2. 4 Effect of mn2+ and Co2+ on 3lp and Ih difference between Zn+ and other divalent ions f ATP (4) Mn+ and Co+ only broaden the peak width The 3P and H spectra were measured for Mn2+ and References Co doping concentrations from 0 to 5.0 x 10-)mol without Mn 2+ compare the 3P and 'H spectra with and [1] Petersen A, Kristensen S, Jacobsen J,et al. 31p-NMR Figures 5 and Co- had a similar result Mn- heasurement of ATP, ADP, 2, 3-diphosphoglycerate and and Co2+ do not cause a chemical shift of the 3lp or h Biophysica Acta, 1990, 1035: 169-174 petra but broaden the peak line width [2] Cohn M, Hughes T. Nuclear magnetic resonance spectra 3 Summary of adenosine di- and triphosphate [J]. J Bio Chemistry 1962,237(1):176-181 (1) The chemical shift of phosphorus nuclei adenosine triphosphate changes as the solution changes. The chemical shift of the B-phosphate is sensitive to pH
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