Isotope Exchange Studies of the Reaction Catalyzed by ATP: Creatine Phosphotransferase

The technique of isotope exchange at equilibrium has been used to determine if the nature of the metal‐nucleotide substrate influences the mechanism of the creatine kinase reaction. Studies of the creatine‐phosphocreatine and ADP‐ATP exchanges in the presence of Mg2+, Ca2+ and Mn2+ showed that the initial rates varied with the total metal ion concentration, that approximately equal rates were obtained for the two exchanges under a particular set of conditions for each metal ion and that these rates were affected to only a small extent by the identity of the metal ion. The creatine‐phosphocreatine and nucleoside diphosphate‐nucleoside triphosphate exchange rates with the cytidine, guanosine and uridine nucleotides in the presence of Mg2+ were of a similar magnitude although very much less than those obtained with the adenine nucleotides. The two exchange rates could be considered as being approximately equal only with the cytidine nucleotides us substrates. The results have been interpreted as indicating that the creatine kinase reaction has a rapid equilibrium, random mechanism, irrespective of the nature of the activating metal ion, when adenine nucleotides are used as substrates and also in the presence of the magnesium complexes of the cytidine nucleotides. No definitive conclusions could be reached about the mechanism from the results obtained with the guanosine and uridine nucleotides. The apparent equilibrium constant of the reaction has been shown to be dependent on the total concentration of metal ions. True equilibrium constants for the reaction in the presence of Mg2+ and Ca2+ have been determined.