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

Hua Deng, Andrzej Lewandowicz, Sean M. Cahill, Richard H. Furneaux, Peter C. Tyler, Mark E. Girvin, Robert H. Callender, Vern L. Schramm

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13 Scopus citations


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·PO 4 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 has been assigned to N-7H. The N-7H resonance is shifted downfield by only ∼1 ppm from its position for ImmH free in aqueous solution, consistent with only a small change in the hydrogen bonding on N-7H upon binding of ImmH to PNP. In contrast, the downfield resonance at 14.9 ppm in the PNP·ImmH· PO4 complex is assigned to N-1H of ImmH by using saturation-transferred NOE measurements on the PNP-ImmH complex. The ∼4 ppm downfield shift of the N-1H resonance from its position for ImmH free in solution suggests that the hydrogen bonding to the N-1H in the complex has a significant contribution to the binding of ImmH to PNP. The crystal structure shows Glu201 is in a direct hydrogen bond with N-1H and to 0-6 through a water bridge. In the complex with 6-thio-ImmH, the N-1H resonance is shifted further downfield by an additional 1.5 ppm to 16.4 ppm, but the relative shift from the value for 6-thio-ImmH free in solution is the same as in the ImmH complex. Since the binding affinity to hPNP for 6-thio-ImmH is decreased 440-fold relative to that for ImmH, the loss in binding energy is primarily due to the hydrogen bond energy loss at the 6-thiol.

Original languageEnglish (US)
Pages (from-to)1980-1987
Number of pages8
Issue number7
StatePublished - Feb 24 2004

ASJC Scopus subject areas

  • Biochemistry


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