Effect of substitution inert metal complexes on calcineurin

As a substitute for M(H2O)2+6, Co(NH3)3+6 was found to activate calcineurin with para-nitrophenyl phosphate as substrate. Kinetics for calcineurin catalyzed hydrolysis of para-nitrophenyl phosphate at pH 7.0 with Mn2+, Mg2+, Co2+, and Co(NH3)3+6 were compared. Although kcat and Km were different with the metals, values of kcat/Km were nearly identical for Mn2+ and Mg2+, but lower for Co2+ and Co(NH3)3+6. The concentration of each metal providing half-maximal activation, designated Kact, was evaluated as 15.9 mM for Co(NH3)3+6, compared to Kact = 0.17 mM for Mn2+ and Co2+ and 6.3 mM for Mg2+, respectively. Comparing kcat/Kcat showed that Co(NH3)3+6 was a 170-fold poorer activator of calcineurin than was Mn2+, but only 1.5-fold poorer than Mg2+. Activation by Co(NH3)3+6 indicated that activation of calcineurin by exogenous metal ions can proceed via an outer coordination sphere reaction mechanism with no requirement for the direct coordination of substrate by metal. Because Co(NH3)3+6 was found to support calcineurin activity, the related compound [Co-(ethylenediamine)3]3+ (or Co(en)3+3) was tested as a possible activator. Co(en)3+3 did not support calcineurin activity but did inhibit calcineurin. Co(en)3+3 showed competitive inhibition kinetics with either Mn2+ or pNPP as the varied ligand and the other at a fixed, subsaturating concentration. Inorganic phosphate was used as a known competitive inhibitor to pNPP (B. L. Martin and D. J. Graves, J. Biol. Chem. 261, 14545-14550, 1986) and showed uncompetitive inhibition with Mn2+ as the varied ligand. These patterns are consistent with the mechanism of ligand binding to calcineurin being ordered with metal preceding substrate. Prior formation of a metal-substrate complex was not required for association with calcineurin.