PMID:11294631
| Citation |
Hyytiä, T, Halonen, P, Salminen, A, Goldman, A, Lahti, R and Cooperman, BS (2001) Ligand binding sites in Escherichia coli inorganic pyrophosphatase: effects of active site mutations. Biochemistry 40:4645-53 |
|---|---|
| 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) 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. |
| Links | |
| Keywords |
Amino Acid Substitution/genetics; Binding Sites/genetics; Binding, Competitive/genetics; Diphosphonates/chemistry; Escherichia coli/enzymology; Escherichia coli/genetics; Hydrogen-Ion Concentration; Ligands; Magnesium/chemistry; Manganese/chemistry; Mutagenesis, Site-Directed; Phosphates/chemistry; Pyrophosphatases/antagonists & inhibitors; Pyrophosphatases/chemistry; Pyrophosphatases/genetics; Static Electricity |
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