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 physi cal approaches. Proton inventory studies of the calcineurin-catalyzed hydrolysis of para-nitrophenyl phosphate were done to probe the role o f proton transfer in the mechanism. Control experiments determined tha t the solvent did not cause the irreversible inactivation of the enzym e and had no effect on the dependence on metal ion or calmodulin. A so lvent isotope effect was observed on the V-max/K-m term, but not the V -max term. The isotope effect was modest with a value of 1.35. Proton inventory data could be fit by multiple parameter sits. The parameter sets yielded fractionation factors of 0.73 for a one-proton transfer o r 0.85 for a two-proton transfer. These values compare to the value of 0.69 for reactions involving a water molecule or hydroxide coordinate d to metal ion. A chemical mechanism consistent with the proton invent ory data and other information about calcineurin catalysis is presente d. The simplest model for catalysis involves a single proton transfer from water coordinated to metal that is reasoned to occur during assoc iation of the substrate with calcineurin. Questions about the reaction intermediate were also addressed. Attempts to monitor a phosphate-wat er exchange reaction with P-31 nuclear magnetic resonance spectroscopy were unsuccessful. Failure to observe an exchange reaction suggests t hat no phosphoryl enzyme is formed during the progress of the reaction . Together these data are explained by a model in which cleavage of th e phosphate ester bond is catalyzed by a water (hydroxide) molecule co ordinated to a divalent metal ion without the formation of a covalent intermediate.