Stoychev Gerasim, Kierdaszuk Borys, Shugar David
Department of Biophysics, Institute of Experimental Physics, University of Warsaw, Poland.
Eur J Biochem. 2002 Aug;269(16):4048-57. doi: 10.1046/j.1432-1033.2002.03097.x.
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.
先前已有关于黄嘌呤（Xan）和黄苷（Xao）作为哺乳动物来源嘌呤核苷磷酸化酶（PNP）底物特性的报道，但仅在一个任意选定的pH值下，且未给出动力学常数。此外，研究未考虑到在生理pH值下，Xao（pKa = 5.7）是单阴离子，而Xan（pKa = 7.7）是中性和单阴离子形式的平衡混合物这一事实。此外，与鸟苷（Guo）、肌苷（Ino）、鸟嘌呤（Gua）和次黄嘌呤（Hx）的单阴离子形式不同，Xao和Xan的单阴离子形式仍是6 - 氧嘌呤。人红细胞和小牛脾脏的PNP对Xao和Xan的最适pH值在5 - 6范围内，而对Guo和Gua以及Ino和Hx的最适pH值在7 - 8范围内。Xao和Xan底物特性的pH依赖性表明中性和阴离子形式均为底物，且明显更倾向于中性形式。6 - 硫代黄嘌呤（pKa = 6.5 ± 0.1）的中性和阴离子形式都是较弱的底物，而2 - 硫代黄嘌呤（pKa = 5.9 ± 0.1）则不是。在pH 5.7时，小牛脾脏PNP将Xao磷解为Xan的反应在83%的转化率时达到平衡，这是由于与逆向合成途径的平衡（平衡常数0.05），而非产物抑制。用砷酸盐替代磷酸导致Xao完全砷解。报道了在几个选定pH值下磷解和逆向合成途径的动力学参数。在pH 5.7时，人源酶对200 μM Xao的磷解受到Guo（IC50 = 10 ± 2 μM）、Hx（IC50 = 7 ± 1 μM）和Gua（IC50 = 4.0 ± 0.2 μM）的抑制。对于Gua，抑制作用表现为竞争性，Ki = 2.0 ± 0.3 μM。相比之下，Xao及其磷解产物（Xan和R1P）对Guo磷解的抑制作用较弱，且Xan不抑制与Gua的逆向反应。提出了哺乳动物PNP对Xan和Xao中性和阴离子形式的可能结合模式。需要注意的是，XMP、Xao和Xan中性和离子形式的结构特性在许多其他酶系统中也至关重要，如肌苷酸脱氢酶、一些核酸聚合酶、咖啡因生物合成和磷酸核糖转移酶。
