Aluminum-adenine nucleotides as alternate substrates for creatine kinase

Aluminum-ATP has previously been shown to be a potent but selective inhibitor of certain kinases (N. C. Furumo and R. E. Viola (1989) Inorg. Chem. 28, 820-823). Because of the selective binding affinity that has been demonstrated, aluminum-ATP was examined as a potential alternate substrate for enzyme-catalyzed phosphoryl-transfer reactions. Of the kinases that have been examined only creatine kinase, which was weakly inhibited by Al-ATP, was found to utilize this metal-nucleotide complex as a substrate. The maximum velocity for this reaction is 0.6% of that observed with Mg-ATP, and a Michaelis constant of 90 microM was measured, which is comparable to that with Mg-ATP. The equilibrium constant for the reaction, measured by phosphorus-31 NMR spectroscopy, is 0.18. This value is indistinguishable from the value determined for the reaction with Mg-ATP as the phosphoryl donor. The rate of interconversion between reactants and products at stoichiometric levels of creatine kinase was found to be quite rapid in the presence of Al3+, indicating that the rate-limiting step in catalytic turnover for this substrate must be a slow rate of substrate binding and/or product release.