Transition State Analysis for Human and Plasmodium falciparum Purine Nucleoside Phosphorylases

Andrzej Lewandowicz, Vern L. Schramm

Research output: Contribution to journalArticle

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Abstract

Recent studies have shown that Plasmodiumfalciparum is sensitive to a purine salvage block at purine nucleoside phosphorylase (PNP) and that human PNP is a target for T-cell proliferative diseases. Specific tight-binding inhibitiors might be designed on the basis of specific PNP transition state structures. Kinetic isotope effects (KIEs) were measured for arsenolysis of inosine catalyzed by P. falciparum and human purine nucleoside phosphorylases. Intrinsic KIEs from [1′-3H]-, [2′-3H]-, [1′-14C]-, [9-15N]-, and [5′- 3H]inosines were 1.184 ± 0.004, 1.031 ± 0.004, 1.002 ± 0.006, 1.029 ± 0.006, and 1.062 ± 0.002 for the human enzyme and 1.116 ± 0.007, 1.036 ± 0.003, 0.996 ± 0.006, 1.019 ± 0.005, and 1.064 ± 0.003 for P. falciparum PNPs, respectively. Analysis of KIEs indicated a highly dissociative D N*AN (SN1) stepwise mechanism with very little leaving group involvement. The near-unity 1′-14C KIEs for both human and P. falciparum PNP agree with the theoretical value for a 1′-14C equilibrium isotope effect for oxacarbenium ion formation when computed at the B1LYP/6-31G(d) level of theory. The 9- 15N KIE for human PNP is also in agreement with theory for equilibrium formation of hypoxanthine and oxacarbenium ion at this level of theory. The 9-15N KIE for P. falciparum PNP shows a constrained vibrational environment around N9 at the transition state. A relatively small β-secondary 2′-3H KIE for both enzymes indicates a 3′-endo conformation for ribose and relatively weak hyperconjugation at the transition state. The large 5′-3H KIE reveals substantial distortion at the 5′-hydroxymethyl group which causes loosening of the C5′-H5′ bonds during the reaction coordinate.

Original languageEnglish (US)
Pages (from-to)1458-1468
Number of pages11
JournalBiochemistry
Volume43
Issue number6
DOIs
StatePublished - Feb 17 2004

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Purine-Nucleoside Phosphorylase
Plasmodium falciparum
Isotopes
Kinetics
Inosine
Ions
Salvaging
Hypoxanthine
Ribose
T-cells
Enzymes
Conformations
T-Lymphocytes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Transition State Analysis for Human and Plasmodium falciparum Purine Nucleoside Phosphorylases. / Lewandowicz, Andrzej; Schramm, Vern L.

In: Biochemistry, Vol. 43, No. 6, 17.02.2004, p. 1458-1468.

Research output: Contribution to journalArticle

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abstract = "Recent studies have shown that Plasmodiumfalciparum is sensitive to a purine salvage block at purine nucleoside phosphorylase (PNP) and that human PNP is a target for T-cell proliferative diseases. Specific tight-binding inhibitiors might be designed on the basis of specific PNP transition state structures. Kinetic isotope effects (KIEs) were measured for arsenolysis of inosine catalyzed by P. falciparum and human purine nucleoside phosphorylases. Intrinsic KIEs from [1′-3H]-, [2′-3H]-, [1′-14C]-, [9-15N]-, and [5′- 3H]inosines were 1.184 ± 0.004, 1.031 ± 0.004, 1.002 ± 0.006, 1.029 ± 0.006, and 1.062 ± 0.002 for the human enzyme and 1.116 ± 0.007, 1.036 ± 0.003, 0.996 ± 0.006, 1.019 ± 0.005, and 1.064 ± 0.003 for P. falciparum PNPs, respectively. Analysis of KIEs indicated a highly dissociative D N*AN (SN1) stepwise mechanism with very little leaving group involvement. The near-unity 1′-14C KIEs for both human and P. falciparum PNP agree with the theoretical value for a 1′-14C equilibrium isotope effect for oxacarbenium ion formation when computed at the B1LYP/6-31G(d) level of theory. The 9- 15N KIE for human PNP is also in agreement with theory for equilibrium formation of hypoxanthine and oxacarbenium ion at this level of theory. The 9-15N KIE for P. falciparum PNP shows a constrained vibrational environment around N9 at the transition state. A relatively small β-secondary 2′-3H KIE for both enzymes indicates a 3′-endo conformation for ribose and relatively weak hyperconjugation at the transition state. The large 5′-3H KIE reveals substantial distortion at the 5′-hydroxymethyl group which causes loosening of the C5′-H5′ bonds during the reaction coordinate.",
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