Conformational states of human purine nucleoside phosphorylase at rest, at work, and with transition state analogues

Achelle A. Edwards, Jeremiah D. Tipton, Michael D. Brenowitz, Mark R. Emmett, Alan G. Marshall, Gary B. Evans, Peter C. Tyler, Vern L. Schramm

Research output: Contribution to journalArticlepeer-review

28 Scopus citations


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 entropie 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.

Original languageEnglish (US)
Pages (from-to)2058-2067
Number of pages10
Issue number9
StatePublished - Mar 9 2010

ASJC Scopus subject areas

  • Biochemistry


Dive into the research topics of 'Conformational states of human purine nucleoside phosphorylase at rest, at work, and with transition state analogues'. Together they form a unique fingerprint.

Cite this