A kinetic method for determining dissociation constants for metal complexes of adenosine 5'-triphosphate and adenosine 5'-diphosphate.

A general kinetic method is described for determining the dissociation constants of metal-ATP complexes that act as inhibitory substrate analogues for any enzyme that utilizes MgATP(2-). The usefulness of the procedure is illustrated by the results obtained from studies of the inhibition of hexokinase by lanthanide-ATP (LnATP) complexes. At relatively low concentrations of Mg2+, these complexes act as linear competitive inhibitors with respect to MgATP(2-). In the presence of higher, fixed concentrations of Mg2+, however, double reciprocal plots of the inhibition by LnATP vs. MgATP are nonlinear, and the data can be used to determine the ratio of the dissociation constants for the LnATP and MgATP complexes. As values are available for the dissociation constant of MgATP under a variety of conditions, that for any LnATP complex can be calculated. The dissociation constant for EuATP at pH 8.0 is 0.16 microM, while that for GdATP is 0.91 microM at pH 6.0, 0.087 microM at pH 7.95, and 1 microM at pH 8.65. Between pH 6 and 8, the ratio of the dissociation constants for GdATP and MgATP(2-) remains constant, and thus, within this range of pH, the lanthanide species involved must be Gd3+ and GdATP-. The method can also be applied to the determination of dissociation constants for inhibitory metal-ADP complexes if MgADP- is used as the variable substrate.