Isotope effects on the mechanism of calcineurin catalysis: kinetic solvent isotope and isotope exchange studies.
The reaction scheme of calcineurin was examined with kinetic and physical approaches. Proton inventory studies of the calcineurin-catalyzed hydrolysis of para-nitrophenyl phosphate were done to probe the role of proton transfer in the mechanism. Control experiments determined that the solvent did not cause the irreversible inactivation of the enzyme and had no effect on the dependence on metal ion or calmodulin. A solvent isotope effect was observed on the Vmax/Km term, but not the Vmax term. The isotope effect was modest with a value of 1.35. Proton inventory data could be fit by multiple parameter sets. The parameter sets yielded fractionation factors of 0.73 for a one-proton transfer or 0.85 for a two-proton transfer. These values compare to the value of 0.69 for reactions involving a water molecule or hydroxide coordinated to metal ion. A chemical mechanism consistent with the proton inventory data and other information about calcineurin catalysis is presented. The simplest model for catalysis involves a single proton transfer from water coordinated to metal that is reasoned to occur during association of the substrate with calcineurin. Questions about the reaction intermediate were also addressed. Attempts to monitor a phosphate-water exchange reaction with 31P nuclear magnetic resonance spectroscopy were unsuccessful. Failure to observe an exchange reaction suggests that no phosphoryl enzyme is formed during the progress of the reaction. Together these data are explained by a model in which cleavage of the phosphate ester bond is catalyzed by a water (hydroxide) molecule coordinated to a divalent metal ion without the formation of a covalent intermediate.
Martin
B L
BL
Department of Biochemistry and Biophysics, Iowa State University, Ames 50011.
Graves
D J
DJ
D000818:Animals; D019703:Calcineurin; D002148:Calmodulin-Binding Proteins / Q000737:chemistry*; D002384:Catalysis; D002417:Cattle; D007700:Kinetics; D010749:Phosphoprotein Phosphatases / Q000737:chemistry*; D012997:Solvents
D002148:Calmodulin-Binding Proteins; D012997:Solvents; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases