Abstract
Previous attempts to elucidate the mechanism of metal-adenosine triphosphate (ATP) binding have resulted in apparent disagreement between the results of two fast reaction techniques, namely, nmr and tempera- ture jump. We have extended the nmr measurements on manganese(II) to low ATP concentrations (ca. 5 X 10-4 M). Competition studies in mixtures of adenosine monophosphate (AMP), ATP, and Mn2 + have also been car- ried out. The results of these measurements confirm the existence of a 1:2 metal-ligand complex at high total ATP. The low concentration studies support the assignment of temperature-jump spectra to the formation of a 1:1 complex. The kinetic scheme at room temperature reconciling the results of the different experimental tech- niques is shown in Scheme I. By using the rate constants for step 1 ;=± 2 obtained by temperature jump, and an estimate of the equilibrium quotient for the metal-independent step 1 ;=i 4, we have been able either to determine or to estimate the remaining rate constants and equilibrium quotients for the metal-dependent steps. These con- stants are shown for 2+. This scheme predicts that the phosphorus and proton magnetic resonance line broaden- ing studies should reach a low concentration asymptote consistent with pathway 1 ;=i 2. This limit was experi- mentally observed in the proton case, where ATP concentrations as low as 5 X 10-4 M could be studied using com- puter enhancement. In the MA2 complex the metal ion simultaneously binds to the phosphate moiety of one nu- cleotide and to the adenine ring nitrogen of the second nucleotide. The MA2 complexes in which the metal ion binds to the N-7 position predominate. At low nucleotide concentration where the MA complex becomes accessi- ble to the nmr, we show that the metal ion is ca. 3.8 Á from the Hs proton. This distance could arise either (1) from the metal ion binding predominantly to the N-7, or (2) from the metal ion being near the Hs but separated from the adenine ring by a coordination shell water molecule. An earlier uv difference study strongly suggests the second alternative.