Ligand Binding Sites inEscherichia coliInorganic Pyrophosphatase: Effects of Active Site Mutations

Abstract

Type I soluble inorganic pyrophosphatases (PPases) are well characterized both structurally and mechanistically. Earlier we measured the effects of active site substitutions on pH−rate profiles for the type I PPases from both Escherichia coli (E-PPase) and Saccharomyces cerevisae (Y-PPase). Here we extend these studies by measuring the effects of such substitutions on the more discrete steps of ligand binding to E-PPase, including (a) Mg²⁺ and Mn²⁺ binding in the absence of added ligand; (b) Mg²⁺ binding in the presence of either P_i or hydroxymethylbisphosphonate (HMBP), a competitive inhibitor of E-PPase; and (c) P_i binding in the presence of Mn²⁺. The active site of a type I PPase has well-defined subsites for the binding of four divalent metal ions (M1−M4) and two phosphates (P1, P2). Our results, considered in light of pertinent results from crystallographic studies on both E-PPase and Y-PPase and parallel functional studies on Y-PPase, allow us to conclude the following: (a) residues E20, D65, D70, and K142 play key roles in the functional organization of the active site; (b) the major structural differences between the product and substrate complexes of E-PPase are concentrated in the lower half of the active site; (c) the M1 subsite is functionally isolated from the rest of the active site; and (d) the M4 subsite is an especially unconstrained part of the active site.