Assignment of Downfield Proton Resonances in Purine Nucleoside Phosphorylase‚Immucillin-H Complex by Saturation-Transferred NOEs †

Purine nucleoside phosphorylase (PNP) catalyzes N-ribosidic bond phosphorolysis in 6-oxypurine nucleosides and deoxynucleosides to form purine and α-d-phosphorylated ribosyl products. The transition state has oxacarbenium ion character with partial positive charge near C-1‘, ionic stabilization from the nearby phosphate anion, and protonation at N-7 of the purine. Immucillin-H (ImmH) has a protonated N-7 and resembles the transition-state charge distribution when N-4‘ is protonated to the cation. It binds tightly to the PNPs with a Kd value 56 pM for human PNP. Previous NMR studies of PNP·ImmH·PO4 have shown that the N-4‘ of bound ImmH is a cation and is postulated to have a significant contribution to its tight binding. Several unassigned downfield proton resonances (>11 ppm) are specific to the PNP·ImmH·PO4 complex, suggesting the existence of strong hydrogen bonds. In this study, two of the proton resonances in this downfield region have been assigned. Using 15N-7-labeled ImmH, a resonance at 12.5 ppm ...