Structure of metal x nucleotide complex in the creatine kinase reaction. A study with diastereomeric phosphorothioate analogs of adenosine di- and triphosphate.

The diastereomers of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) and adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) were tested as substrates for creatine kinase in the presence of different activating divalent metal cations. In the presence of Mg2+, the Rp diastereomers of both ATP alpha S and ATP beta S are the preferred substrates, whereas in the presence of Cd2+, the Sp diastereomers are preferred. In the reverse reaction the Rp isomer of ADP alpha S is the better substrate in the presence of Mg2+ while the Sp isomer is preferred in the presence of Cd2+. In the presence of Mg2+, only the Rp isomer of ATP beta S is synthesized from prochiral ADP beta S while the Sp isomer is synthesized predominantly in the presence of Cd2+. In the presence of Ca2+, Mn2+, and Co2+, loss of substrate specificity is observed. These results are explained on the basis of the observation that Mg2+ prefers to coordinate to oxygen and Cd2+ to sulfur in these phosphorothioate analogs (Jaffe, E. K., and Cohn, M. (1978) J. Biol. Chem. 253, 4823-4825). Thus, the metal ion appears to be bound to both the alpha- and beta-phosphates at some stage of the reaction. The interpretation is that the substrate binds as the lambda, beta, gamma-bidentate MgATP chelate. It can then undergo either nucleophilic substitution at the gamma-phosphorus followed by migration of the metal to yield the alpha, beta MgADP complex or metal migration followed by subsequent phosphoryl transfer. The product of the reaction is delta, alpha, beta-bidentate MgADP. The different reaction routes are discussed.