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 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.
Hengge
A C
AC
Institute for Enzyme Research, University of Wisconsin, 1710 University Avenue, Madison, Wisconsin 53705, USA.
Martin
B L
BL
D000818:Animals; D019703:Calcineurin; D002147:Calmodulin / Q000737:chemistry*; D002148:Calmodulin-Binding Proteins / Q000737:chemistry*; D002417:Cattle; D006868:Hydrolysis; D007554:Isotopes; D008345:Manganese / Q000737:chemistry*; D009596:Nitrophenols / Q000737:chemistry*; D009943:Organophosphorus Compounds / Q000737:chemistry*; D010749:Phosphoprotein Phosphatases / Q000737:chemistry*
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D007554:Isotopes; D009596:Nitrophenols; D009943:Organophosphorus Compounds; C008644:nitrophenylphosphate; D008345:Manganese; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases