Catalysis by Escherichia coli Inorganic Pyrophosphatase: pH and Mg2+ Dependence

Abstract

Steady-state rates of PP_i hydrolysis by Escherichia coli inorganic pyrophosphatase (E-PPase) were measured as a function of magnesium pyrophosphate (substrate) and free Mg²⁺ ion (activator) in the pH range 6.0−10.0. Computer fitting of hydrolysis data in combination with direct measurements of Mg²⁺ binding to enzyme has resulted in a model that quantitatively accounts for our results. The major features of this model are the following: (a) E-PPase catalysis proceeds both with three and with four (and possibly with five) Mg²⁺ ions per active site; (b) catalysis requires both an essential base and an essential acid, and the pK_as of these groups are modulated by the stoichiometry of bound Mg²⁺; and (c) the four-metal route predominates for concentrations of free Mg²⁺ >0.2 mM. The model straightforwardly accounts for the apparent linkage between increased pK_a of an essential base and activity requirements for higher Mg²⁺ concentration observed for several active site variants. Microscopic rate constants for overall catalysis of PP_i−P_i equilibration were determined at pH 6.5−9.3 by combined analysis of enzyme-bound PP_i formation and rates of PP_i hydrolysis, PP_i synthesis, and P_i−H₂O oxygen exchange. The catalytic activity of E-PPase at saturating substrate increases toward PP_i hydrolysis and decreases toward PP_i synthesis and P_i−H₂O oxygen exchange with increasing pH. These changes are mainly due to an increased rate of dissociation of the second released P_i and a decreased rate of enzyme-bound PP_i synthesis from enzyme-bound P_i, respectively, as the pH is raised.