| Abstract
| - Activation of calcineurin by Mn2+ and Mg2+ was compared using a heavy atom isotope analogueof the substrate p-nitrophenyl phosphate (pNPP). Heavy atom isotope effects were measured for Mg2+activation and compared to published results of the isotope effects with Mn2+ as the activating metal.Isotope effects were measured for the kinetic parameter Vmax/Km at 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 nitrogenatom in the nitrophenol leaving group [15(V/K)]. The isotope effects increased in magnitude upon changingfrom 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 for18(V/K)nonbridge is 0.9910 (±0.0003) at pH 7.0. As with Mn2+, the 18(V/K)nonbridge isotope effect indicatedthat the dianion was the substrate for catalysis, and that a dissociative transition state was operative forthe phosphoryl transfer. Comparison to results for Mn2+ activation suggested that chemistry was morerate-limiting with Mg2+ than with Mn2+. Changing the activating metal concentration showed oppositetrends with increasing Mg2+ increasing the commitment factor and seemingly making the chemistry lessrate-limiting. The influence of viscosity was evaluated as well to gauge the role of chemistry. The activationof calcineurin-catalyzed hydrolysis of pNPP1 by Mg2+ or Mn2+ at pH 7.0 was compared in the presenceof viscogens, glycerol and poly(ethylene glycol). Increasing glycerol caused different effects with thetwo activators. With Mn2+ as the activator, calcineurin activity showed a normal response with kcat andkcat/Km decreasing with viscosity. There was an inverse response with Mg2+ as the activator as values ofkcat/Km increased with viscosity. From values of the normalized kcat/Km with Mn2+, the chemistry wasfound to be partially rate-limiting, consistent with previous heavy atom isotope studies (). The effectobserved for Mg2+ seems consistent with a change in the rate-limiting step for the two different metalsat pH 7.0.
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