31P nuclear magnetic resonance spectra of the thiophosphate analogues of adenine nucleotides; effects of pH and Mg2+ binding.

The 31P nuclear magnetic resonance (NMR) spectra of the adenine nucleotide thio analogues, AMPS, ADPalphaS, ADPbetaS, ATPalphaS, ATPbetaS, and ATPgammaS, have been studied. Of primary interest were the increased sensitivity of chemical shifts to protonation and to magnesium binding of these analogues compared with the corresponding effects on AMP, ADP, and ATP. The usefulness of the characteristic NMR parameters of the thio analogues as probes in enzymatic reactions is discussed. The A2 diastereoisomers of ADPalphaS and ATPalphaS and the A and B isomers of ATPbetaS were enzymatically synthesized and the diasterioisomers of ADPalphaS and ATPbetaS were distinguished by their 31P NMR parameters. The stereospecificity of the enzymatic reactions involving the thio analogues of nucleotides can therefore be determined by 31P NMR. The difficulty involved in assigning phosphate ligands of Mg in MgADP and MgATP and their analogues on the basis of the magnitude of chemical shift changes (deltadelta) induced by Mg binding upon each 31P is discussed in the context of the anomalies in deltadelta of each 31P observed upon protonation of the terminal phosphate group. It is concluded that chemical shift data cannot yield unequivocal information concerning the absolute structure of metal complexes of nucleotides but can be used to monitor changes in metal chelation, for example, upon binding to enzyme.