Metal Ion Binding to Adenosine Triphosphate. III. A Kinetic Analysis

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 temperature 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 carried out. The results of these measurements confirm the existence of a 1:2 metalligand 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 techniques 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 ⇌ 4, we have been able either to determine or to estimate the remaining rate constants and equilibrium quotients for the metal-dependent steps. These constants are shown for Mn2+. This scheme predicts that the phosphorus and proton magnetic resonance line broadening studies should reach a low concentration asymptote consistent with pathway 1 ⇌ 2. This limit was experimentally observed in the proton case, where ATP concentrations as low as 5 × 10−4 M could be studied using computer 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 H8 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 H8 but separated from the adenine ring by a coordination shell water molecule. An earlier uv difference study strongly suggests the second alternative. © 1968, American Chemical Society. All rights reserved.