A1 Refereed original research article in a scientific journal
Ligand binding sites in Escherichia coli inorganic pyrophosphatase: Effects of active site mutations
Authors: Hyytia T, Halonen P, Salminen A, Goldman A, Lahti R, Cooperman BS
Publisher: AMER CHEMICAL SOC
Publication year: 2001
Journal name in source: BIOCHEMISTRY
Journal acronym: BIOCHEMISTRY-US
Volume: 40
Issue: 15
First page : 4645
Last page: 4653
Number of pages: 9
ISSN: 0006-2960
DOI: https://doi.org/10.1021/bi010049x
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, D6, 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.
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, D6, 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.
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