Nuclear Magnetic Resonance Study of Metal-Ion Binding to Adenosine Triphosphate. I. 31P Studies

Abstract

Measurements of the ³¹P NMR of the α, β, and γ phosphates of adenosine triphosphate (ATP) have been made for Mn²⁺—, Ni²⁺—, Co²⁺—, and Cu²⁺—ATP complexes. In order to explain the metal‐ion—phosphate interaction, measurements were made at different values of f≡[M²⁺]/[ATP] between 0° and 95°C. The metal ions broadened the ³¹P NMR, described by a decrease in the observed value of T₂; reduced T₁, the spin—lattice relaxation time; and in the case of CoATP at high temperatures, shifted the resonance to lower fields by an amount Δω_M because of the transferred isotropic hyperfine coupling A. For Mn²⁺ and Ni²⁺ at all temperatures and CoATP at lower temperatures, T₂ was determined by the chemical exchange time τ_M of the metal—phosphate bonds, and all three phosphates had the same widths. For CoATP at higher temperatures, the resonances were exchange narrowed and ${\text{1/T}}_2{\mathrm{\approx f\tau }}_{\mathrm{M}}{\mathrm{\Delta \omega }}_{\mathrm{M}}^2$ . In agreement with the shift measurements ${\text{1/T}}_2{|}_{\gamma }{\text{>1/T}}_2{|}_{\beta }{\text{>1/T}}_2{|}_{\alpha }$ . The values of A from the shifts were (in megacycles per second): A_γ=5.4, A_β=4.5, and A_α=3.6 which were slightly larger than the values in Co₃(PO₄)₂ crystals of 3.3. The large values of A in ATP show that the Co²⁺ is bound all the time to the phosphates. The T₁ values for MnATP complexes are determined by dipolar interactions of the form ${\text{1/T}}_1{\mathrm{\hspace{0.17em}\propto \hspace{0.17em}(\mu /r}}_3{)}^2{\tau }_c$ . At room temperatures $T_1{|}_{\gamma }{\mathrm{=T}}_1{|}_{\beta }=\frac{3}{4}{T}_1{|}_{\alpha }$ , which implies a very small difference in the distance of the metal ion from all three ³¹P nuclei. At higher temperature all three were equal. Assuming the phosphorous—metal distance of 3.3 Å, τ_c is determined to be ∼1×10⁻⁹ sec, which is about four times larger than the same correlation time for the water protons. From the high‐temperature MnATP linewidths, which were all equal, we conclude that the hyperfine coupling with all three ³¹P nuclei is >0.6 times the value of A observed by Mays in LiMnPO₄ crystals, once again showing that the binding to all three phosphates is approximately complete and approximately the same.