Yeast Inorganic Pyrophosphatase

The kinetics of the Mg2+ activation of crystalline yeast inorganic pyrophosphatase have been thoroughly investigated from pH 7.40 to pH 9.05 using a sensitive isotope assay in order to determine the role of the divalent metal ion activator in the reaction. A computer program has been devised for calculating the concentration of each of the various components of the complex equilibrium involving Mg2+ and inorganic pyrophosphate (PPi). The reaction rate was measured over a wide range of Mg2+ and PPi concentrations, and the concentration dependence of the measured rate was kinetically analyzed by a computerized algorithm for nonlinear regression. The computer analysis included the testing of several plausible kinetic models for goodness of fit to the data, and determination of best values of kinetic parameters for the various models. The simplest kinetic model which provides a good fit to all of the data involves binding of free Mg2+ by the enzyme followed by binding of PPi ligands. Both MgPPi and Mg2PPi are substrates; at pH 7.40 the latter is hydrolyzed 22% as rapidly as the former. Free PPi is bound but is not hydrolyzed at a significant rate. On the basis of these kinetic studies we propose two roles for the metal ion in this reaction: activation of the enzyme to a form which binds substrate, and formation of substrate by complexing with PPi. These roles are entirely consistent with our previously reported equilibrium binding studies of this enzyme.