Structural investigations of the Mg.ATP complex at the active site of porcine adenylate kinase using phosphorothioate analogs and electron paramagnetic resonance of Mn(II) with chiral 17O-labelled ATP analogs

The interaction between ATP, divalent metal ions and the active site of adenylate kinase from muscle has been studied by two different methods. No reversal of the relative reaction rates of the two diastereomers of adenosine 5′‐([α‐thio]triphosphate) was observed on changing the metal ion from Mg2+ to Cd2+, suggesting that the α‐phosphate group is not coordinated to the divalent metal ion in the enzyme · metal · ATP complex. This interpretation is confirmed by the lack of influence of 17O incorporated into the α‐phosphate group of ATP on the electron paramagnetic resonance (EPR) spectrum of Mn2+ at the active site of the enzyme. The specificity for the Sp diastereomer thus appears to be due to an interaction of the enzyme active site with the pro‐R oxygen of the α‐phosphate group. In contrast, the specificity of adenylate kinase for the diastereomers of adenosine 5′([β‐thio]triphosphate) is reversed from Sp to Rp on replacing Mg2+ by Cd2+, indicating an interaction of the pro‐R oxygen of the β‐phosphate group of ATP with the metal ion in the enzyme · metal · ATP complex. In agreement with this, ATP which is regio‐specifically labelled with 17O on the β‐phosphate group causes a line‐broadening of the EPR spectrum of Mn2+ at the active site, demonstrating coordination of at least one of the β‐phosphate oxygens with the metal. Using stereospecifically labelled [β‐17O]ATPs, it was found that (Rp)‐[β‐17O]ATP but not (Sp)‐[β‐17O]ATP caused a line broadening in the Mn2+ EPR spectrum similar to that seen with regio‐labelled [β‐17O]ATP, further indicating that the pro‐R oxygen of the β‐phosphate group of ATP is coordinated to the metal ion at the active site of adenylate kinase. These results lead to a value of 0.22–0.25 mT for the superhyperfine coupling constant of a single 17O‐Mn2+ interaction in a manganese‐ATP‐enzyme complex. ATP labelled with 17O on the γ‐phosphate group has no effect on the spectrum of Mn2+ at the active site, leading to the conclusion that Mg · ATP exists predominantly as a β‐monodentate complex at the active site of adenylate kinase. From the effect of H217O on the EPR spectrum of Mn2+ in the Mn · ATP complex at the active site it appears most likely that three, four or five water molecules are bound to the metal ion