Isotope effect studies on the calcineurin phosphoryl-transfer reaction: transition state structure and effect of calmodulin and Mn2+

Biochemistry. 1997 Aug 19;36(33):10185-91. doi: 10.1021/bi9706374.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Calcineurin
  • Calmodulin / chemistry*
  • Calmodulin-Binding Proteins / chemistry*
  • Cattle
  • Hydrolysis
  • Isotopes
  • Manganese / chemistry*
  • Nitrophenols / chemistry*
  • Organophosphorus Compounds / chemistry*
  • Phosphoprotein Phosphatases / chemistry*

Substances

  • Calmodulin
  • Calmodulin-Binding Proteins
  • Isotopes
  • Nitrophenols
  • Organophosphorus Compounds
  • nitrophenylphosphate
  • Manganese
  • Calcineurin
  • Phosphoprotein Phosphatases