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Enhancement of the rate of pyrophosphate hydrolysis by nonenzymatic catalysts and by inorganic pyrophosphatase
Authors:Stockbridge Randy B  Wolfenden Richard
Institution:Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Abstract: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 (PPi) 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 MgPPi2? 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 PPi hydrolysis by reducing the enthalpy of activation. Mg2+ increased the rate of PPi hydrolysis by both increasing the entropy of activation and reducing the enthalpy of activation.
Keywords:Calorimetry  Enzyme Catalysis  Enzyme Mechanisms  Metals  Phosphatase  Pyrophosphatase  Rate Enhancement
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