Tryptophan-free human PNP reveals catalytic site interactions

Mahmoud Ghanem, Suwipa Saen-oon, Nickolay Zhadin, Corin Wing, Sean M. Cahill, Steven D. Schwartz, Robert Callender, Vern L. Schramm

Research output: Contribution to journalArticle

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Abstract

Human purine nucleoside Phosphorylase (PNP) is a homotrimer, containing three nonconserved tryptophan residues at positions 16, 94, and 178, all remote from the catalytic site. The Tip residues were replaced with Tyr to produce Trp-free PNP (Leuko-PNP). Leuko-PNP showed near-normal kinetic properties. It was used (1) to determine the tautomeric form of guanine that produces strong fluorescence when bound to PNP, (2) for thermodynamic binding analysis of binary and ternary complexes with substrates, (3) in temperature-jump perturbation of complexes for evidence of multiple conformational complexes, and (4) to establish the ionization state of a catalytic site tyrosine involved in phosphate nucleophile activation. The 13C NMR spectrum of guanine bound to Leuko-PNP, its fluorescent properties, and molecular orbital electronic transition analysis establish that its fluorescence originates from the lowest singlet excited state of the N1H, 6-keto, N7H guanine tautomer. Binding of guanine and phosphate to PNP and Leuko-PNP are random, with decreased affinity for formation of ternary complexes. Pre-steady-state kinetics and temperature-jump studies indicate that the ternary complex (enzyme-substrate- phosphate) forms in single binding steps without kinetically significant protein conformational changes as monitored by guanine fluorescence. Spectral changes of Leuko-PNP upon phosphate binding establish that the hydroxyl of Tyr88 is not ionized to the phenolate anion when phosphate is bound. A loop region (residues 243-266) near the purine base becomes highly ordered upon substrate/inhibitor binding. A single Tip residue was introduced into the catalytic loop of Leuko-PNP (Y249W-Leuko-PNP) to determine effects on catalysis and to introduce a fluorescence catalytic site probe. Although Y249W-Leuko-PNP is highly fluorescent and catalytically active, substrate binding did not perturb the fluorescence. Thermodynamic boxes, constructed to characterize the binding of phosphate, guanine, and hypoxanthine to native, Leuko-, and Y249W-Leuko-PNPs, establish that Leuko-PNP provides a versatile protein scaffold for introduction of specific Trp catalytic site probes.

Original languageEnglish (US)
Pages (from-to)3202-3215
Number of pages14
JournalBiochemistry
Volume47
Issue number10
DOIs
StatePublished - Mar 11 2008

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Purine-Nucleoside Phosphorylase
Tryptophan
Catalytic Domain
Guanine
Phosphates
Fluorescence
Substrates
Thermodynamics
Nucleophiles
Hypoxanthine
Kinetics
Temperature
Molecular orbitals
Catalysis
Excited states
Scaffolds
Hydroxyl Radical

ASJC Scopus subject areas

  • Biochemistry

Cite this

Ghanem, M., Saen-oon, S., Zhadin, N., Wing, C., Cahill, S. M., Schwartz, S. D., ... Schramm, V. L. (2008). Tryptophan-free human PNP reveals catalytic site interactions. Biochemistry, 47(10), 3202-3215. https://doi.org/10.1021/bi702491d

Tryptophan-free human PNP reveals catalytic site interactions. / Ghanem, Mahmoud; Saen-oon, Suwipa; Zhadin, Nickolay; Wing, Corin; Cahill, Sean M.; Schwartz, Steven D.; Callender, Robert; Schramm, Vern L.

In: Biochemistry, Vol. 47, No. 10, 11.03.2008, p. 3202-3215.

Research output: Contribution to journalArticle

Ghanem, M, Saen-oon, S, Zhadin, N, Wing, C, Cahill, SM, Schwartz, SD, Callender, R & Schramm, VL 2008, 'Tryptophan-free human PNP reveals catalytic site interactions', Biochemistry, vol. 47, no. 10, pp. 3202-3215. https://doi.org/10.1021/bi702491d
Ghanem M, Saen-oon S, Zhadin N, Wing C, Cahill SM, Schwartz SD et al. Tryptophan-free human PNP reveals catalytic site interactions. Biochemistry. 2008 Mar 11;47(10):3202-3215. https://doi.org/10.1021/bi702491d
Ghanem, Mahmoud ; Saen-oon, Suwipa ; Zhadin, Nickolay ; Wing, Corin ; Cahill, Sean M. ; Schwartz, Steven D. ; Callender, Robert ; Schramm, Vern L. / Tryptophan-free human PNP reveals catalytic site interactions. In: Biochemistry. 2008 ; Vol. 47, No. 10. pp. 3202-3215.
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N2 - Human purine nucleoside Phosphorylase (PNP) is a homotrimer, containing three nonconserved tryptophan residues at positions 16, 94, and 178, all remote from the catalytic site. The Tip residues were replaced with Tyr to produce Trp-free PNP (Leuko-PNP). Leuko-PNP showed near-normal kinetic properties. It was used (1) to determine the tautomeric form of guanine that produces strong fluorescence when bound to PNP, (2) for thermodynamic binding analysis of binary and ternary complexes with substrates, (3) in temperature-jump perturbation of complexes for evidence of multiple conformational complexes, and (4) to establish the ionization state of a catalytic site tyrosine involved in phosphate nucleophile activation. The 13C NMR spectrum of guanine bound to Leuko-PNP, its fluorescent properties, and molecular orbital electronic transition analysis establish that its fluorescence originates from the lowest singlet excited state of the N1H, 6-keto, N7H guanine tautomer. Binding of guanine and phosphate to PNP and Leuko-PNP are random, with decreased affinity for formation of ternary complexes. Pre-steady-state kinetics and temperature-jump studies indicate that the ternary complex (enzyme-substrate- phosphate) forms in single binding steps without kinetically significant protein conformational changes as monitored by guanine fluorescence. Spectral changes of Leuko-PNP upon phosphate binding establish that the hydroxyl of Tyr88 is not ionized to the phenolate anion when phosphate is bound. A loop region (residues 243-266) near the purine base becomes highly ordered upon substrate/inhibitor binding. A single Tip residue was introduced into the catalytic loop of Leuko-PNP (Y249W-Leuko-PNP) to determine effects on catalysis and to introduce a fluorescence catalytic site probe. Although Y249W-Leuko-PNP is highly fluorescent and catalytically active, substrate binding did not perturb the fluorescence. Thermodynamic boxes, constructed to characterize the binding of phosphate, guanine, and hypoxanthine to native, Leuko-, and Y249W-Leuko-PNPs, establish that Leuko-PNP provides a versatile protein scaffold for introduction of specific Trp catalytic site probes.

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