title: Kinetics of phosphorolysis of 3-(beta-D-ribofuranosyl)adenine and 3-(beta-D-ribofuranosyl)hypoxanthine, non-conventional substrates of purine-nucleoside phosphorylase.
abstract: The properties of two non-conventional substrates of the calf-spleen and Escherichia coli purine nucleoside phosphorylases (PNP), 3-(beta-D-ribofuranosyl)adenine (RibfAde) and 3-(beta-D-ribofuranosyl)hypoxanthine (RibfHyp), are described. In contrast to Ado, RibfAde is a substrate for the mammalian enzyme. With the calf enzyme, the pseudo-first-order rate constants (Vmax/K(m)) for phosphorolysis of RibfAde and RibfHyp are 3% and 13%, respectively, that for phosphorolysis of Ino, while for E. coli PNP the corresponding values are 22% and 30%, respectively. The Michaelis constants (K(m)) for RibfAde were 800 microM (calf PNP) and 150 microM (E. coli PNP). For RibfHyp, the corresponding K(m) values were 220 microM and 260 microM. Two well-characterized inhibitors of calf spleen PNP [9-(2-fluoro-3,4-dihydroxybutyl)guanine] and E. coli PNP (formycin A) were found to inhibit phosphorolysis of RibfAde and RibfHyp with the same inhibition constants as for Ino. Moreover, the inhibition was competitive, which indicates that phosphorolysis of 3-beta-nucleosides occurs at the same active site as for the natural substrate Ino. In particular, the substrate properties of both 3-beta-nucleosides are consistent with their binding to the enzyme in the conformation anti to the imidazole ring about the glycosidic bond, which is superimposable on the structure of natural 9-beta-nucleosides in the conformation anti to the pyrimidine ring. The results are examined in relation to present concepts regarding the binding of substrates and inhibitors at the active site(s) of these enzymes.
authors.affiliation: Department of Biophysics, University of Warsaw, Poland.
authors.forename: A
authors.initials: A
authors.lastname: Bzowska
authors.forename: E
authors.initials: E
authors.lastname: Kulikowska
authors.forename: N E
authors.initials: NE
authors.lastname: Poopeiko
authors.forename: D
authors.initials: D
authors.lastname: Shugar
mesh_terms: D000241:Adenosine / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D006868:Hydrolysis; D007288:Inosine / Q000031:analogs & derivatives* / Q000378:metabolism; D007700:Kinetics; D011485:Protein Binding; D011683:Purine-Nucleoside Phosphorylase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; C062751:3-isoadenosine; D007288:Inosine; C062282:3-isoinosine; D011683:Purine-Nucleoside Phosphorylase; D000241:Adenosine