Comparison of the reaction progress of calcineurin with Mn2+ and Mg2+

Activation of calcineurin by Mn2+ and Mg2+ was compared using a heavy atom isotope analogue of the substrate p-nitrophenyl phosphate (pNPP), Heavy ato m isotope effects were measured for Mg2+ activation and compared to publish ed results of the isotope effects with Mn2+ as the activating metal. Isotop e effects were measured for the kinetic parameter V-max/K-m at the nonbridg ing oxygen atoms [(18)(V/K)(nonbridge)]; at the position of bond cleavage i n 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 i n magnitude upon changing from an optimal pH to a nonoptimal pH; the (18)(V /K)(bridge) effect increased from 1.0154 (+/-0.0007) to 1.0198 (+/-0.0002), and the (15)(V/K) effect: increased from 1.0018 (+/-0.0002) to 1.0021 (+/- 0.0003). The value for (18)(V/K)(nonbridge) is 0.9910 (+/-0.0003) at pH 7.0 . As with Mn2+, the (18)(V/K)(nonbridge) isotope effect indicated that the dianion was the substrate for catalysis, and that a dissociative transition state was operative for the phosphoryl transfer. Comparison to results for Mn2+ activation suggested that chemistry was more rate-limiting with Mg2than with Mn2+. Changing the activating metal concentration showed opposite trends with increasing Mg2+ increasing the commitment factor and seemingly making the chemistry less rate-limiting. The influence of viscosity was ev aluated as well to gauge the role of chemistry. The activation of calcineur in-catalyzed hydrolysis of pNPP(1) by Mg2+ or Mn2+ at pH 7.0 was compared i n the presence of viscogens, glycerol and poly(ethylene glycol). Increasing glycerol caused different effects with the two activators. With Mn2+ as th e activator, calcineurin activity showed a normal response with k(cat) and k(cat)/K-m decreasing with viscosity. There was an inverse response with Mg 2+ as the activator as values of k(cat)/K-m increased with viscosity. From values of the normalized k(cat)/K-m with Mn2+, the chemistry was found to b e partially rate-limiting, consistent with previous heavy atom isotope stud ies (22). The effect observed for Mg2+ seems consistent with a change in th e rate-limiting step for the two different metals at pH 7.0.