Comparison of the Reaction Progress of Calcineurinwith Mn²⁺ and Mg²⁺

Authors

B. L. Martin
L. A. Jurado
Alvan C. Hengge, Utah State UniversityFollow

Document Type

Article

Journal/Book Title

Biochemistry

Publication Date

1999

Volume

38

First Page

3386

Last Page

3392

Abstract

Activation of calcineurin by Mn²⁺ and Mg²⁺ was compared using a heavy atom isotope analogue of 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 V_max/K_m 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 nitrogen atom in the nitrophenol leaving group [¹⁵(V/K)]. The isotope effects increased in 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 Mg²⁺ than 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 evaluated as well to gauge the role of chemistry. The activation of calcineurin-catalyzed hydrolysis of pNPP¹ by Mg²⁺ or Mn²⁺ at pH 7.0 was compared in the presence of viscogens, glycerol and poly(ethylene glycol). Increasing glycerol caused different effects with the two activators. With Mn²⁺ as the activator, calcineurin activity showed a normal response with k_cat and k_cat/K_m decreasing with viscosity. There was an inverse response with Mg²⁺ 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 be partially rate-limiting, consistent with previous heavy atom isotope studies (22). The effect observed for Mg²⁺ seems consistent with a change in the rate-limiting step for the two different metals at pH 7.0.

Recommended Citation

B. L. Martin, L. A. Jurado and A. C. Hengge “Comparison of the Reaction Progress of Calcineurin with Mn2+ and Mg2+.” Biochemistry, 1999, 38, 3386 –3392.