Enhancement of the rate of pyrophosphate hydrolysis by nonenzymatic catalysts and by inorganic pyrophosphatase

To estimate the proficiency of inorganic pyrophosphatase as a catalyst, ³¹P NMR was used to determine rate constants and thermodynamics of activation for the spontaneous hydrolysis of inorganic pyrophosphate (PP_i) in the presence and absence of Mg²⁺ at elevated temperatures. These values were compared with rate constants and activation parameters determined for the reaction catalyzed by Escherichia coli inorganic pyrophosphatase using isothermal titration calorimetry. At 25 °C and pH 8.5, the hydrolysis of MgPP_i²⁻ proceeds with a rate constant of 2.8 × 10⁻¹⁰ s⁻¹, whereas E. coli pyrophosphatase was found to have a turnover number of 570 s⁻¹ under the same conditions. The resulting rate enhancement (2 × 10¹²-fold) is achieved entirely by reducing the enthalpy of activation (ΔΔH^‡ = −16.6 kcal/mol). The presence of Mg²⁺ ions or the transfer of the substrate from bulk water to dimethyl sulfoxide was found to increase the rate of pyrophosphate hydrolysis by as much as ∼10⁶-fold. Transfer to dimethyl sulfoxide accelerated PP_i hydrolysis by reducing the enthalpy of activation. Mg²⁺ increased the rate of PP_i hydrolysis by both increasing the entropy of activation and reducing the enthalpy of activation.