Ligand binding sites in Escherichia coli inorganic pyrophosphatase: effects of active site mutations.
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) Mg(2+) and Mn(2+) binding in the absence of added ligand; (b) Mg(2+) 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 Mn(2+). 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.
Hyytiä
T
T
Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6323, USA.
Halonen
P
P
Salminen
A
A
Goldman
A
A
Lahti
R
R
Cooperman
B S
BS
D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive / Q000235:genetics; D004164:Diphosphonates / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008274:Magnesium / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000737:chemistry; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics*; D055672:Static Electricity
D004164:Diphosphonates; D008024:Ligands; D010710:Phosphates; C029285:hydroxymethanediphosphonic acid; D008345:Manganese; D011755:Pyrophosphatases; D008274:Magnesium