Isotope effect studies on the calcineurin phosphoryl-transfer reaction: Transition state structure and effect of calmodulin and Mn2+

The hydrolysis of p-nitrophenyl phosphate (pNPP) catalyzed by calcineurin has been studied by measurement of heavy-atom isotope effects in the substrate, The isotope effects were measured al the nonbridging oxygen atoms [(18)(V/K)(nonbridge)], at the position of bond cleavage in the bridging oxygen atom [(18)(V/K)(bridge)], and at the nitrogen atom in the nitrophenol leaving group [(15)(V/K)]. The isotope effects increased in magnitude upon moving from the pH optimum of 7.0 to 8.5; (18)(V/K)(bridge) increased from 1.0072 to 1.0115, and (15)(V/K) from 1.0006 to 1.0014. The value for (18)(V/K)(nonbridge) is 0.9942 at pH 8.5. These data are consistent with P-O bond cleavage being partially rate-limiting at the pH optimum and more SO at the higher pH. The (18)(V/K)(nonbridge) isotope effect indicates that the dianion is the substrate for catalysis, and a dissociative transition state is operative for phosphoryl transfer, Increasing the concentration of the activating metal ion Mn2+ at pH 7.0 from 1 mM to 5 mM increases the magnitude of the isotope effects by an amount similar to that observed with the shift in pH from 7.0 to 8.5, indicative of a change in the commitment factor in the kinetic mechanism so as to make the chemical step more rate-limiting.