Probing the mechanism of purine nucleoside phosphorylase by steady-state kinetic studies and ligand binding characterization determined by fluorimetric titrations

Biochimica Et Biophysica Acta
Beata Wielgus-Kutrowska, A Bzowska

Abstract

Reversible reaction catalyzed by trimeric purine nucleoside phosphorylase (PNP) from Cellulomonas sp. with typical and non-typical substrates, including product inhibition patterns of both reaction directions, and interactions of the enzyme with bisubstrate analogue inhibitors, were investigated by the steady-state kinetic methods and fluorimetric titrations. The ligand chromophores exist most probably as neutral species, and not N(1)-H monoanions, in the complex with PNP, as shown by determination of inhibition constants vs. pH. This supports the mechanism in which hydrogen bond interaction of N(1)-H with Glu204 is crucial in the catalytic process. Stoichiometry of ligand binding, with possible exception of hypoxanthine, is three molecules per enzyme trimer. Kinetic experiments show that in principle the Michaelis-Menten model could not properly describe the reaction. However, this model seems to hold for certain experimental conditions. Data presented here are supported by earlier findings obtained by means of fluorimetric titrations and protective effects of ligands on thermal inactivation of the enzyme. All results are consistent with the following mechanism for trimeric PNPs: (i) random binding of substrates, (ii) potent b...Continue Reading

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Citations

Aug 16, 2014·Molecular BioSystems·Jacek WierzchowskiDavid Shugar
Jan 6, 2016·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Alicja Stachelska-WierzchowskaBeata Wielgus-Kutrowska
Mar 3, 2009·Bioorganic & Medicinal Chemistry·Grzegorz MedzaDavid Shugar
Jul 1, 2008·Protein Expression and Purification·Katarzyna BreerAgnieszka Bzowska
Apr 22, 2015·Journal of Biomolecular Structure & Dynamics·Vishakha Karnawat, Mrinalini Puranik
Jan 26, 2010·The Journal of Physical Chemistry. B·Eun Jung ShimStacey D Wetmore
Dec 31, 2009·The Journal of Physical Chemistry. B·Jennifer L Przybylski, Stacey D Wetmore
Feb 14, 2008·Biochemistry·Mahmoud GhanemVern L Schramm

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