Isotope Effect Studies on the Calcineurin Phosphoryl- TransferReaction: Transition State Structure and Effect of Calmodulin and Mn+2
Document Type
Article
Journal/Book Title
Biochemistry
Publication Date
1997
Volume
36
First Page
10185
Last Page
10191
Abstract
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 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 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.
Recommended Citation
A. C. Hengge and B. L. Martin. “Isotope Effect Studies on the Calcineurin Phosphoryl- Transfer Reaction: Transition State Structure and Effect of Calmodulin and Mn+2,” Biochemistry, 1997, 36, 10185-10191.
