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

Research output: Contribution to journalArticle

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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

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Purine-Nucleoside Phosphorylase
Trichomonas vaginalis
nucleoside phosphotransferase
Adenosine
immucillin A
Purine Nucleosides
Salvaging
Static Electricity
Isotopes
Hydroxyl Radical
Cations
Hydrogen
Electrostatics
Infrared spectroscopy
Spectrum Analysis
Hydrogen bonds

ASJC Scopus subject areas

  • Biochemistry

Cite this

Inhibition and structure of Trichomonas vaginalis purine nucleoside phosphorylase with picomolar transition state analogues. / Rinaldo-Matthis, Agnes; Wing, Corin; Ghanem, Mahmoud; Deng, Hua; Wu, Peng; Gupta, Arti; Tyler, Peter C.; Evans, Gary B.; Furneaux, Richard H.; Almo, Steven C.; Wang, Ching C.; Schramm, Vern L.

In: Biochemistry, Vol. 46, No. 3, 23.01.2007, p. 659-668.

Research output: Contribution to journalArticle

Rinaldo-Matthis, A, Wing, C, Ghanem, M, Deng, H, Wu, P, Gupta, A, Tyler, PC, Evans, GB, Furneaux, RH, Almo, SC, Wang, CC & Schramm, VL 2007, 'Inhibition and structure of Trichomonas vaginalis purine nucleoside phosphorylase with picomolar transition state analogues', Biochemistry, vol. 46, no. 3, pp. 659-668. https://doi.org/10.1021/bi061515r
Rinaldo-Matthis, Agnes ; Wing, Corin ; Ghanem, Mahmoud ; Deng, Hua ; Wu, Peng ; Gupta, Arti ; Tyler, Peter C. ; Evans, Gary B. ; Furneaux, Richard H. ; Almo, Steven C. ; Wang, Ching C. ; Schramm, Vern L. / Inhibition and structure of Trichomonas vaginalis purine nucleoside phosphorylase with picomolar transition state analogues. In: Biochemistry. 2007 ; Vol. 46, No. 3. pp. 659-668.
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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 {\AA} 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 {\AA}. 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.",
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AU - Rinaldo-Matthis, Agnes

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AU - Deng, Hua

AU - Wu, Peng

AU - Gupta, Arti

AU - Tyler, Peter C.

AU - Evans, Gary B.

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N2 - 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.

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