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.

The previous finding that 7-methylinosine (m7Ino) and 7-methylguanosine (m7Guo) are excellent, as well as fluorescent, substrates for calf spleen purine nucleoside phosphorylase has been extended to include a series of 7-alkylguanosines with higher alkyl groups (ethyl, propyl, butyl, isopropyl, isobutyl, benzyl). All of these are good substrates, with increases in Km compensated for by corresponding increases in Vmax, and excluding the ring N-7 as a binding site. Both m1Ino and m1Guo are neither substrates nor inhibitors of this enzyme, implicating the ring N-1 as a binding site. Included also are guanosine analogues with C-8 substituents (alpha-hydroxyisopropyl, methyl, bromo, chloro, amino) with known populations of syn and anti conformers about the glycosidic bond; while these did not unequivocally point to involvement of only one form, efficiency of phosphorolysis appeared to be correlated with a conformation in the anti region. The influence of various substituents is also considered in relation to steric and electronic effects. Kinetic parameters for all the foregoing have been determined fluorimetrically and/or spectrophotometrically from initial velocities and/or continuous monitoring of phosphorolysis including Ki values for inhibition by the base moieties. Furthermore, kinetic data demonstrated that, in striking contrast to the mammalian enzyme, both m1Ino and m1Guo are almost as good substrates as the parent nucleosides for a bacterial (Escherichia coli) enzyme, suggesting that the ring N-1 is not a binding site for the latter.