NICOTINAMIDE RIBOSIDE, AN UNUSUAL, NON-TYPICAL, SUBSTRATE OF PURIFIEDPURINE-NUCLEOSIDE PHOSPHORYLASES

Nicotinamide 1-beta-D-riboside (Nir), the cationic, reducible moiety o f the coenzyme NAD(+), has been confirmed as an unusual substrate for purified purine-nucleoside phosphorylase (PNP) from a mammalian source (calf spleen). It is also a substrate of the enzyme from Escherichia coli. The K-m values at pH 7, 1.48 mM and 0.62 mM, respectively, were 1-2 orders of magnitude higher than for the natural substrate inosine, but the V-max values were comparable, 96% and 35% that for Ino. The p seudo first-order rate constants, V-max/K-m, were 1.1% and 2.5% for th e calf spleen and E. coli enzymes. The aglycon, nicotinamide, was neit her a substrate nor an inhibitor of PNP. Nir was a weak inhibitor of i nosine phosphorolysis catalyzed by both enzymes, with K-i values close to the K-m for its phosphorolysis, consistent with simple competitive inhibition; this was further confirmed by Dixon plots. Phosphorolysis of the fluorescent positively charged substrate 7-methylguanosine was also inhibited in a competitive manner by both Ino and Nir. Phosphoro lysis of Nir by both enzymes was inhibited competitively by several sp ecific inhibitors of calf spleen and E. coli PNP, with K-i values simi lar to those for inhibition of other natural substrates. The pH depend ence of the kinetic constants for the phosphorolysis of Nir and of a v ariety of other substrates, was extensively investigated, particularly in the alkaline pH range, where Nir exhibited abnormally high substra te activity relative to the reduced reaction rates of both enzymes tow ards other anionic or neutral substrates. The overall results are disc ussed in relation to present concepts regarding binding and phosphorol ysis of substrates by PNP based on crystallographic data of enzyme-inh ibitor complexes, and current studies on enzymatic and nonenzymatic me chanisms of the cleavage of the Nir glycosidic bond.