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

摘要: 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; 8.6 pM). ImmH binds with larger entropic penalty than DATMe-ImmH, chemically more flexible inhibitor. The testable hypothesis that PNP conformational states are relaxed (dynamic) despite tighter ImmH. conformations probed by peptide amide deuterium exchange (HDX) using liquid chromatography high-resolution Fourier transform ion cyclotron resonance mass spectrometry sedimentation rates. Catalytically equilibrating Michaelis complexes (PNP·PO4·inosine ↔ PNP·Hx·R-1-P) inhibited (PNP·PO4·DATMe-ImmH PNP·PO4·ImmH) show protection from HDX at 9, 13, 15 sites per subunit relative resting (PNP·PO4) in extended incubations. PNP·PO4·ImmH complex compact (by rate) other complexes. kinetic analysis of liga...