Active site contacts in the purine nucleoside phosphorylase-hypoxanthine complex by NMR and ab initio calculations

Hua Deng, Sean M. Cahill, José Luis Abad, Andrzej Lewandowicz, Robert Callender, Vern L. Schramm, Roger A. Jones

Research output: Contribution to journalArticle

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Abstract

Hypoxanthine (Hx) with specific 15N labels has been used to probe hydrogen-bonding interactions with purine nucleoside phosphorylase (PNP) by NMR spectroscopy. Hx binds to human PNP as the N-7H tautomer, and the N-7H 1H and 15N chemical shifts are located at 13.9 and 156.5 ppm, respectively, similar to the solution values. In contrast, the 1H and 15N chemical shifts of N-1H in the PNP·Hx complex are shifted downfield by 3.5 and 7.5 ppm to 15.9 and 178.8 ppm, respectively, upon binding. Thus, hydrogen bonding at N-1H is stronger than at N-7H in the complex. Ab initio chemical shift calculations on model systems that simulate Hx in solution and bound to PNP are used to interpret the NMR data. The experimental N-7H chemical shift changes are caused by competing effects of two active site contacts. Hydrogen bonding of Glu201 to N-1H causes upfield shifts of the N-7H group, while the local hydrogen bond (C=O to N-7H from Asn243) causes downfield shifts. The observed N-7H chemical shift can be reproduced by a hydrogen bond distance ∼0.13 Å shorter (but within experimental error) of the experimental value found in the X-ray crystal structure of the bovine PNP·Hx complex. The combined use of NMR and ab initio chemical shift computational analysis provides a novel approach to understand enzyme-ligand interactions in PNP, a target for anticancer agents. This approach has the potential to become a high-resolution tool for structural determination.

Original languageEnglish (US)
Pages (from-to)15966-15974
Number of pages9
JournalBiochemistry
Volume43
Issue number50
DOIs
StatePublished - Dec 21 2004

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Purine-Nucleoside Phosphorylase
Hypoxanthine
Chemical shift
Catalytic Domain
Hydrogen Bonding
Nuclear magnetic resonance
Hydrogen bonds
Hydrogen
Antineoplastic Agents
Magnetic Resonance Spectroscopy
X-Rays
Ligands
Nuclear magnetic resonance spectroscopy
Labels
Enzymes
Crystal structure
X rays

ASJC Scopus subject areas

  • Biochemistry

Cite this

Active site contacts in the purine nucleoside phosphorylase-hypoxanthine complex by NMR and ab initio calculations. / Deng, Hua; Cahill, Sean M.; Abad, José Luis; Lewandowicz, Andrzej; Callender, Robert; Schramm, Vern L.; Jones, Roger A.

In: Biochemistry, Vol. 43, No. 50, 21.12.2004, p. 15966-15974.

Research output: Contribution to journalArticle

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title = "Active site contacts in the purine nucleoside phosphorylase-hypoxanthine complex by NMR and ab initio calculations",
abstract = "Hypoxanthine (Hx) with specific 15N labels has been used to probe hydrogen-bonding interactions with purine nucleoside phosphorylase (PNP) by NMR spectroscopy. Hx binds to human PNP as the N-7H tautomer, and the N-7H 1H and 15N chemical shifts are located at 13.9 and 156.5 ppm, respectively, similar to the solution values. In contrast, the 1H and 15N chemical shifts of N-1H in the PNP·Hx complex are shifted downfield by 3.5 and 7.5 ppm to 15.9 and 178.8 ppm, respectively, upon binding. Thus, hydrogen bonding at N-1H is stronger than at N-7H in the complex. Ab initio chemical shift calculations on model systems that simulate Hx in solution and bound to PNP are used to interpret the NMR data. The experimental N-7H chemical shift changes are caused by competing effects of two active site contacts. Hydrogen bonding of Glu201 to N-1H causes upfield shifts of the N-7H group, while the local hydrogen bond (C=O to N-7H from Asn243) causes downfield shifts. The observed N-7H chemical shift can be reproduced by a hydrogen bond distance ∼0.13 {\AA} shorter (but within experimental error) of the experimental value found in the X-ray crystal structure of the bovine PNP·Hx complex. The combined use of NMR and ab initio chemical shift computational analysis provides a novel approach to understand enzyme-ligand interactions in PNP, a target for anticancer agents. This approach has the potential to become a high-resolution tool for structural determination.",
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N2 - Hypoxanthine (Hx) with specific 15N labels has been used to probe hydrogen-bonding interactions with purine nucleoside phosphorylase (PNP) by NMR spectroscopy. Hx binds to human PNP as the N-7H tautomer, and the N-7H 1H and 15N chemical shifts are located at 13.9 and 156.5 ppm, respectively, similar to the solution values. In contrast, the 1H and 15N chemical shifts of N-1H in the PNP·Hx complex are shifted downfield by 3.5 and 7.5 ppm to 15.9 and 178.8 ppm, respectively, upon binding. Thus, hydrogen bonding at N-1H is stronger than at N-7H in the complex. Ab initio chemical shift calculations on model systems that simulate Hx in solution and bound to PNP are used to interpret the NMR data. The experimental N-7H chemical shift changes are caused by competing effects of two active site contacts. Hydrogen bonding of Glu201 to N-1H causes upfield shifts of the N-7H group, while the local hydrogen bond (C=O to N-7H from Asn243) causes downfield shifts. The observed N-7H chemical shift can be reproduced by a hydrogen bond distance ∼0.13 Å shorter (but within experimental error) of the experimental value found in the X-ray crystal structure of the bovine PNP·Hx complex. The combined use of NMR and ab initio chemical shift computational analysis provides a novel approach to understand enzyme-ligand interactions in PNP, a target for anticancer agents. This approach has the potential to become a high-resolution tool for structural determination.

AB - Hypoxanthine (Hx) with specific 15N labels has been used to probe hydrogen-bonding interactions with purine nucleoside phosphorylase (PNP) by NMR spectroscopy. Hx binds to human PNP as the N-7H tautomer, and the N-7H 1H and 15N chemical shifts are located at 13.9 and 156.5 ppm, respectively, similar to the solution values. In contrast, the 1H and 15N chemical shifts of N-1H in the PNP·Hx complex are shifted downfield by 3.5 and 7.5 ppm to 15.9 and 178.8 ppm, respectively, upon binding. Thus, hydrogen bonding at N-1H is stronger than at N-7H in the complex. Ab initio chemical shift calculations on model systems that simulate Hx in solution and bound to PNP are used to interpret the NMR data. The experimental N-7H chemical shift changes are caused by competing effects of two active site contacts. Hydrogen bonding of Glu201 to N-1H causes upfield shifts of the N-7H group, while the local hydrogen bond (C=O to N-7H from Asn243) causes downfield shifts. The observed N-7H chemical shift can be reproduced by a hydrogen bond distance ∼0.13 Å shorter (but within experimental error) of the experimental value found in the X-ray crystal structure of the bovine PNP·Hx complex. The combined use of NMR and ab initio chemical shift computational analysis provides a novel approach to understand enzyme-ligand interactions in PNP, a target for anticancer agents. This approach has the potential to become a high-resolution tool for structural determination.

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