Inhibition and structure of Trichomonas vaginalis purine nucleoside phosphorylase with picomolar transition state analogues

Agnes Rinaldo-Matthis, Corin Wing, Mahmoud Ghanem, Hua Deng, Peng Wu, Arti Gupta, Peter C. Tyler, Gary B. Evans, Richard H. Furneaux, Steven C. Almo, Ching C. Wang, Vern L. Schramm

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28 Scopus citations

Abstract

Trichomonas vaginalis is a parasitic protozoan purine auxotroph possessing a unique purine salvage pathway consisting of a bacterial type purine nucleoside phosphorylase (PNP) and a purine nucleoside kinase. Thus, T. vaginalis PNP (TvPNP) functions in the reverse direction relative to the PNPs in other organisms. Immucillin-A (ImmA) and DADMe-Immucillin-A (DADMe-ImmA) are transition state mimics of adenosine with geometric and electrostatic features that resemble early and late transition states of adenosine at the transition state stabilized by TvPNP. ImmA demonstrates slow-onset tight-binding inhibition with TvPNP, to give an equilibrium dissociation constant of 87 pM, an inhibitor release half-time of 17.2 min, and a Km/Kd ratio of 70,100. DADMe-ImmA resembles a late ribooxacarbenium ion transition state for TvPNP to give a dissociation constant of 30 pM, an inhibitor release half-time of 64 min, and a Km/Kd ratio of 203,300. The tight binding of DADMe-ImmA supports a late SN1 transition state. Despite their tight binding to TvPNP, ImmA and DADMe-ImmA are weak inhibitors of human and P. falciparum PNPs. The crystal structures of the TvPNP·ImmA·PO 4 and TvPNP·DADMe-ImmA-PO4 ternary complexes differ from previous structures with substrate analogues. The tight binding with DADMe-ImmA is in part due to a 2.7 Å ionic interaction between a PO 4 oxygen and the N1′ cation of the hydroxypyrrolidine and is weaker in the TvPNP·ImmA·PO4 structure at 3.5 Å. However, the TvPNP·ImmA·PO4 structure includes hydrogen bonds between the 2′-hydroxyl and the protein that are not present in TvPNP·DADMe-ImmA·PO4. These structures explain why DADMe-ImmA binds tighter than ImmA. Immucillin-H is a 12 nM inhibitor of TvPNP but a 56 pM inhibitor of human PNP. And this difference is explained by isotope-edited difference infrared spectroscopy with [6-18O]ImmH to establish that O6 is the keto tautomer in TvPNP·ImmH·PO 4, causing an unfavorable leaving-group interaction.

Original languageEnglish (US)
Pages (from-to)659-668
Number of pages10
JournalBiochemistry
Volume46
Issue number3
DOIs
StatePublished - Jan 23 2007

ASJC Scopus subject areas

  • Biochemistry

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    Rinaldo-Matthis, A., Wing, C., Ghanem, M., Deng, H., Wu, P., Gupta, A., Tyler, P. C., Evans, G. B., Furneaux, R. H., Almo, S. C., Wang, C. C., & Schramm, V. L. (2007). Inhibition and structure of Trichomonas vaginalis purine nucleoside phosphorylase with picomolar transition state analogues. Biochemistry, 46(3), 659-668. https://doi.org/10.1021/bi061515r