Jeremiah Tipton - Conformational States of Human Purine Nucleoside Phosphorylase at Rest, at Work and with Transition State Analogues

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      Publication Details (including relevant citation   information):

      Edwards, A.A.; Tipton, J.D.; Brenowitz, M.D.;   Emmett, M.R. Marshall, A.G; Evans, G.B.; Tyler, P.C.; Schramm,   V.L. Biochemistry, 49 (9), 2058-2067,   (2010)


      Human purine nucleoside phosphorylase (PNP) is a homotrimer   binding tightly to the transition state analogues Immucillin-H   (ImmH; Kd = 56 pM) and   DATMe-ImmH-Immucillin-H (DATMe-ImmH; Kd = 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·PO4·inosine ↔ PNP·Hx·R-1-P) and   inhibited complexes (PNP·PO4·DATMe-ImmH and   PNP·PO4·ImmH) show protection from HDX at 9, 13, and   15 sites per subunit relative to resting PNP (PNP·PO4)   in extended incubations. The PNP·PO4·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.

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