| Abstract: | An equilibrium mixture of highly enriched 18(O)]Pi (represents the mixture of 18(O)4]Pi, 18(O)3]Pi, 18(O)2]Pi as represented in the figures, unless otherwise specified), alpha-D-ribose 1-16(O)]phosphate, and hypoxanthine plus inosine was equilibrated with calf spleen purine-nucleoside phosphorylase (EC 2.4.2.1). The 31P NMR spectrum clearly indicated the formation of alpha-D-ribose 1-18(O)4]-phosphate and of 16(O)]Pi. Incubation for the same time span in the absence of alpha-D-ribose 1-phosphate left the 18(O)4]Pi isotopic distribution unchanged. The results clearly demonstrated that the C--O bond of alpha-D-ribose 1-phosphate is cleaved in the enzymatic reaction. It is unlikely that the enzyme catalyzes the exchange of oxygen between Pi and H2O. Several possible mechanistic pathways are ruled out by the results, which demand attack by a phosphate oxygen at the anomeric C-1' atom. |
