Purine nucleoside phosphorylase (PNP), from calf spleen, human erythrocytes and E. coli have been examined with regard to structural requirements of substrates and inhibitors. Kinetic parameters (Km, Vmax/Km) for a variety of N(1) and/or N(7)-methylated analogues of guanosine, inosine and adenosine have been evaluated for all three enzym es. The substrate and/or inhibitor properties of purine riboside, 1,6-dihydropurine riboside, some deazapurine nucleosides: 3-deaza- and 7-deazainosine, 1,3-dideazapurine riboside (ribobenzimidazole), and a variety of acyclonu cleosides, have been determined with mammalian and bacterial enzymes. Overall results indicate distinct similarities of kinetic properties and structural requirements of the two mammalian enzymes, although there are some differences as well. The N(1) and O6 of the purine ring are necessary for substrate-inhibitor activity and constitute a binding site for the mammalian (but not the bacterial) enzymes. Moreover, nucleosides lacking the N(3) undergo phosphorolysis and those lacking N(7) are inhibitors (but not substrates). Methylation of the ring N(7) leads to two overlapping effects: labilization of the glycosidic bond, and impediment to proton ation at this site by the enzyme, a postulated prerequisite for enzymatic phosphorolysis. It is proposed that a histidine interacts with N(1) as a don or and O6 as an acceptor. Alternatively N(1)−H and C(2)−NH2, may serve as donors for hydrogen bonds with a glutam ate residue.
The less specific E. coli enzyme phosphorolyses all purine ring modified nucleosides but 7-deazainosine which is only an inhibitor. On the other hand, the bacterial enzyme exhibits decreased activity towards N(7)-methylated nucleosides and lack of affinity for a majority of the tested acyclonu cleoside inhibitors of the mammalian enzymes. The foregoing results underline the fundamental differences between mammalian and bacterial enzymes, including variations in the binding sites for the purine ring.
Purine nucleoside phosphorylase. Structure-activity relationships for substrate and inhibitor properties of N-1-, N-7-, and C-8-substituted analogues; differentiation of mammalian and bacterial enzymes with N-1-methylinosine and guanosine.
