title: Substrate binding in protein-tyrosine phosphatase-like inositol polyphosphatases.
abstract: Protein-tyrosine phosphatase-like inositol polyphosphatases are microbial enzymes that catalyze the stepwise removal of one or more phosphates from highly phosphorylated myo-inositols via a relatively ordered pathway. To understand the substrate specificity and kinetic mechanism of these enzymes we have determined high resolution, single crystal, x-ray crystallographic structures of inactive Selenomonas ruminantium PhyA in complex with myo-inositol hexa- and pentakisphosphate. These structures provide the first glimpse of a myo-inositol polyphosphatase-ligand complex consistent with its known specificity and reveal novel features of the kinetic mechanism. To complement the structural studies, fluorescent binding assays have been developed and demonstrate that the K(d) for this enzyme is several orders of magnitude lower than the K(m). Together with rapid kinetics data, these results suggest that the protein tyrosine phosphatase-like inositol polyphosphatases have a two-step, substrate-binding mechanism that facilitates catalysis.
authors.affiliation: Departments of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Alberta, Canada T1K 3M4.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Gruninger
authors.affiliation: Departments of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Alberta, Canada T1K 3M4.
authors.forename: Selina
authors.initials: S
authors.lastname: Dobing
authors.affiliation: Departments of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Alberta, Canada T1K 3M4.
authors.forename: Adam D
authors.initials: AD
authors.lastname: Smith
authors.affiliation: Departments of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Alberta, Canada T1K 3M4.
authors.forename: Lisza M
authors.initials: LM
authors.lastname: Bruder
authors.affiliation: Departments of Biological Sciences, University of Lethbridge, Lethbridge, Alberta, Canada T1K 3M4.
authors.forename: L Brent
authors.initials: LB
authors.lastname: Selinger
authors.affiliation: Departments of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Alberta, Canada T1K 3M4.
authors.forename: Hans-Joachim
authors.initials: HJ
authors.lastname: Wieden
authors.affiliation: Departments of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Alberta, Canada T1K 3M4. Electronic address: steven.mosimann@uleth.ca.
authors.forename: Steven C
authors.initials: SC
authors.lastname: Mosimann
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D007295:Inositol Phosphates / Q000737:chemistry*; D007700:Kinetics; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry*; D011485:Protein Binding; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry*; D020357:Selenomonas / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D007295:Inositol Phosphates; D010744:Phosphoric Monoester Hydrolases; C021822:myo-inositol-1 (or 4)-monophosphatase; D017027:Protein Tyrosine Phosphatases