| Calcineurin is a cytosolic protein serine phosphatase. It is a heterodimeric metalloenzyme, with intrinsically bound zinc and iron found in the active site, but also requires the presence of exogenous divalent metal ions for full activity. In this work, the participation of metal ions in calcineurin catalysis was studied using kinetic approaches. Previous work showed that transition metals and Mg2+ could activate. Co(NH3) 63+ is a substitution inert metal complex and is able to substitute for Mg2+ in numerous enzyme systems and functions in outer-sphere interactions. Co(NH3)63+ was found to activate calcineurin at a level comparable to Mg2+. A structurally related complex, Co(en)33+, was also studied but failed to activate calcineurin. Instead, it was found to act as a competitive inhibitor compared to exogenous metal ion. This complex has higher conformational rigidity and the failure to activate provides information about the size of the enzyme pocket for the activating metal ion. The binding order for activating metal ion and substrate was evaluated to be an ordered reaction mechanism with metal ion binding first. Activation with Co(NH 3)63+ indicates that an outer sphere mechanism was used by calcineurin. The metal ion likely participates in the reaction mechanism through an outer-sphere interaction, such as protein donation or hydrogen bonding.; The dependencies of the measured parameters for the hydrolysis of substrate on pH and temperature were examined to further probe the determinants of the role of exogenous metal ion. Thermodynamic studies were consistent with transition metal ions activating calcineurin via the stabilization of the transition state predominantly through an entropic contribution in contrast to the activation by Mg2+ causing stabilization predominantly through an enthalpic contribution. Intrinsic metal ion is also involved in catalysis although the mechanistic aspects are not well characterized. The oxidation state of iron in the active site has been postulated to regulate the participation of iron (Fe3+ or Fe2+) in catalysis. Using recombinant calcineurin, no effect of ascorbic acid or dithiothreitol (DTT) were observed. These data suggest that intrinsic iron does not need to be in a reduced state for activity. |
