Xanthosine and xanthine. Substrate properties with purine nucleoside phosphorylases, and relevance to other enzyme systems.
Substrate properties of xanthine (Xan) and xanthosine (Xao) for purine nucleoside phosphorylases (PNP) of mammalian origin have been reported previously, but only at a single arbitrarily selected pH and with no kinetic constants. Additionally, studies have not taken into account the fact that, at physiological pH, Xao (pKa = 5.7) is a monoanion, while Xan (pKa = 7.7) is an equilibrium mixture of the neutral and monoanionic forms. Furthermore the monoanionic forms, unlike those of guanosine (Guo) and inosine (Ino), and guanine (Gua) and hypoxanthine (Hx), are still 6-oxopurines. The optimum pH for PNP from human erythrocytes and calf spleen with both Xao and Xan is in the range 5-6, whereas those with Guo and Gua, and Ino and Hx, are in the range 7-8. The pH-dependence of substrate properties of Xao and Xan points to both neutral and anionic forms as substrates, with a marked preference for the neutral species. Both neutral and anionic forms of 6-thioxanthine (pKa = 6.5 +/- 0.1), but not of 2-thioxanthine (pKa = 5.9 +/- 0.1), are weaker substrates. Phosphorolysis of Xao to Xan by calf spleen PNP at pH 5.7 levels off at 83% conversion, due to equilibrium with the reverse synthetic pathway (equilibrium constant 0.05), and not by product inhibition. Replacement of Pi by arsenate led to complete arsenolysis of Xao. Kinetic parameters are reported for the phosphorolytic and reverse synthetic pathways at several selected pH values. Phosphorolysis of 200 micro m Xao by the human enzyme at pH 5.7 is inhibited by Guo (IC50 = 10 +/- 2 micro m), Hx (IC50 = 7 +/- 1 micro m) and Gua (IC50 = 4.0 +/- 0.2 micro m). With Gua, inhibition was shown to be competitive, with Ki = 2.0 +/- 0.3 micro m. By contrast, Xao and its products of phosphorolysis (Xan and R1P), were poor inhibitors of phosphorolysis of Guo, and Xan did not inhibit the reverse reaction with Gua. Possible modes of binding of the neutral and anionic forms of Xan and Xao by mammalian PNPs are proposed. Attention is directed to the fact that the structural properties of the neutral and ionic forms of XMP, Xao and Xan are also of key importance in many other enzyme systems, such as IMP dehydrogenase, some nucleic acid polymerases, biosynthesis of caffeine and phosphoribosyltransferases.
Stoychev
Gerasim
G
Department of Biophysics, Institute of Experimental Physics, University of Warsaw, Poland.
Kierdaszuk
Borys
B
Shugar
David
D
D000818:Animals; D002417:Cattle; D004798:Enzymes / Q000378:metabolism; D004912:Erythrocytes / Q000201:enzymology; D006147:Guanine / Q000378:metabolism; D006151:Guanosine / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D019271:Hypoxanthine / Q000378:metabolism; D007288:Inosine / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D015394:Molecular Structure; D011485:Protein Binding; D011683:Purine-Nucleoside Phosphorylase / Q000378:metabolism*; D012263:Ribonucleosides / Q000737:chemistry / Q000378:metabolism*; D013154:Spleen / Q000201:enzymology; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D019820:Xanthine / Q000737:chemistry / Q000378:metabolism*; D014970:Xanthines / Q000378:metabolism
D004798:Enzymes; D012263:Ribonucleosides; D014970:Xanthines; D006151:Guanosine; D019820:Xanthine; C054700:6-thioxanthine; D019271:Hypoxanthine; D007288:Inosine; D006147:Guanine; C005893:xanthosine; D011683:Purine-Nucleoside Phosphorylase