Loop-tryptophan human purine nucleoside phosphorylase reveals submillisecond protein dynamics

Mahmoud Ghanem, Nickolay Zhadin, Robert Callender, Vern L. Schramm

Research output: Contribution to journalArticle

13 Scopus citations

Abstract

Human PNP is a homotrimer containing three tryptophan residues at positions 16, 94, and 178, all remote from the catalytic site. The catalytic sites of PNP are located near the subunit - subunit interfaces where F159 is a catalytic site residue donated from an adjacent subunit. F159 covers the top (β) surface of the ribosyl group at the catalytic site. QM/MM calculations of human PNP have shown that F159 is the center of the most mobile region of the protein providing access to the substrate in the active site. F159 is also the key residue in a cluster of hydrophobic residues that shield catalytic site ligands from bulk solvent. Trp-free human PNP (Leuko-PNP) was previously engineered by replacing the three Trp residues of native PNP with Tyr. From this active construct, a single Trp residue was placed in the catalytic site loop (F159W-Leuko-PNP) as a reporter group for the ribosyl region of the catalytic site. The F159W-Leuko-PNP fluorescence is red shifted compared to native PNP, suggesting a solvent-exposed Trp residue. Upon ligand binding (hypoxanthine), the 3-fold fluorescence quench confirms conformational packing of the catalytic site pocket hydrophobic cluster. F159W-Leuko-PNP has an onenzyme thermodynamic equilibrium constant (Keq) near unity in the temperature range between 20 and 30 °C and nonzero enthalpic components, making it suitable for laser-induced T-jump analyses. T-jump relaxation kinetics of F159W-Leuko-PNP in equilibrium with substrates and/or products indicate the conformational equilibria of at least two ternary complex intermediates in the nano- to millisecond time scale (1000-10000 s-1) that equilibrate prior to the slower chemical step (∼200 s-1). F159W-Leuko-PNP provides a novel protein platform to investigate the protein conformational dynamics occurring prior to transition state formation.

Original languageEnglish (US)
Pages (from-to)3658-3668
Number of pages11
JournalBiochemistry
Volume48
Issue number16
DOIs
StatePublished - Apr 28 2009

ASJC Scopus subject areas

  • Biochemistry

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