title: Conformational states of human purine nucleoside phosphorylase at rest, at work, and with transition state analogues.
abstract: Human purine nucleoside phosphorylase (PNP) is a homotrimer binding tightly to the transition state analogues Immucillin-H (ImmH; K(d) = 56 pM) and DATMe-ImmH-Immucillin-H (DATMe-ImmH; K(d) = 8.6 pM). ImmH binds with a larger entropic penalty than DATMe-ImmH, a chemically more flexible inhibitor. The testable hypothesis is that PNP conformational states are more relaxed (dynamic) with DATMe-ImmH, despite tighter binding than with ImmH. PNP conformations are probed by peptide amide deuterium exchange (HDX) using liquid chromatography high-resolution Fourier transform ion cyclotron resonance mass spectrometry and by sedimentation rates. Catalytically equilibrating Michaelis complexes (PNP.PO(4).inosine <--> PNP.Hx.R-1-P) and inhibited complexes (PNP.PO(4).DATMe-ImmH and PNP.PO(4).ImmH) show protection from HDX at 9, 13, and 15 sites per subunit relative to resting PNP (PNP.PO(4)) in extended incubations. The PNP.PO(4).ImmH complex is more compact (by sedimentation rate) than the other complexes. HDX kinetic analysis of ligand-protected sites corresponds to peptides near the catalytic sites. HDX and sedimentation results establish that PNP protein conformation (dynamic motion) correlates more closely with entropy of binding than with affinity. Catalytically active turnover with saturated substrate sites causes less change in HDX and sedimentation rates than binding of transition state analogues. DATMe-ImmH more closely mimics the transition of human PNP than does ImmH and achieves strong binding interactions at the catalytic site while causing relatively modest alterations of the protein dynamic motion. Transition state analogues causing the most rigid, closed protein conformation are therefore not necessarily the most tightly bound. Close mimics of the transition state are hypothesized to retain enzymatic dynamic motions related to transition state formation.
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
authors.forename: Achelle A
authors.initials: AA
authors.lastname: Edwards
authors.forename: Jeremiah D
authors.initials: JD
authors.lastname: Tipton
authors.forename: Michael D
authors.initials: MD
authors.lastname: Brenowitz
authors.forename: Mark R
authors.initials: MR
authors.lastname: Emmett
authors.forename: Alan G
authors.initials: AG
authors.lastname: Marshall
authors.forename: Gary B
authors.initials: GB
authors.lastname: Evans
authors.forename: Peter C
authors.initials: PC
authors.lastname: Tyler
authors.forename: Vern L
authors.initials: VL
authors.lastname: Schramm
mesh_terms: D000577:Amides / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D041961:Deuterium Exchange Measurement; D019277:Entropy; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011684:Purine Nucleosides / Q000737:chemistry* / Q000378:metabolism; D011683:Purine-Nucleoside Phosphorylase / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D011744:Pyrimidinones / Q000737:chemistry* / Q000378:metabolism; D013816:Thermodynamics; D014867:Water / Q000737:chemistry / Q000378:metabolism
chemical_list: D000577:Amides; D010455:Peptides; D011684:Purine Nucleosides; D011744:Pyrimidinones; D014867:Water; C113101:forodesine; D011683:Purine-Nucleoside Phosphorylase