Ligand binding sites in Escherichia coli inorganic pyrophosphatase: Effects of active site mutations

Type I soluble inorganic pyrophosphatases (PPases) are well characterized b oth structurally and mechanistically. Earlier we measured the effects of ac tive 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 Mn 2+ binding in the absence of added ligand; (b) Mg2+ binding in the presence of either P-i or hydroxymethylbisphosphonate (HMBP), a competitive inhibit or 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 divale nt 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 conclu de the following: (a) residues E20, D6, D70, and K142 play key roles in the functional organization of the active site; (b) the major structural diffe rences between the product and substrate complexes of E-PPase are concentra ted in the lower half of the active site; (c) the M1 subsite is functionall y isolated from the rest of the active site; and (d) the M4 subsite is an e specially unconstrained part of the active site.