Probing the mechanism of purine nucleoside phosphorylase by steady-state kinetic studies and ligand binding characterization determined by fluorimetric titrations.
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 binding and slow release of some reaction products leading to the circumstances that the chemical step is not the slowest one and that rapid-equilibrium assumptions do not hold, (iii) a dual role of phosphate--a substrate and also a reaction modifier.
Wielgus-Kutrowska
Beata
B
Department of Biophysics, Institute of Experimental Physics, Warsaw University, Zwirki i Wigury 93, 02-089 Warsaw, Poland.
Bzowska
Agnieszka
A
D002384:Catalysis; D006147:Guanine / Q000031:analogs & derivatives / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008024:Ligands; D010766:Phosphorylation; D011485:Protein Binding; D011683:Purine-Nucleoside Phosphorylase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D017186:Titrimetry
D008024:Ligands; D006147:Guanine; C008450:7-methylguanine; D011683:Purine-Nucleoside Phosphorylase