Properties of two unusual, and fluorescent, substrates of purine-nucleoside phosphorylase: 7-methylguanosine and 7-methylinosine

Abstract

The properties of two unusual substrates of calf spleen purine-nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1), 7-methylguanosine and 7-methylinosine, are described. The corresponding bases, 7-methylguanine and 7-methylhypoxanthine, are neither substrates in the reverse, synthetic reaction, nor inhibitors of the phosphorolysis reaction. Both nucleosides exhibit fluorescence, which disappears on cleavage of the glycosidic bond, providing a new convenient procedure for continuous fluorimetric assay of enzymatic activity. For 7-methylguanosine at neutral pH and 25°C, V max = 3.3 μmol/min per unit enzyme and K m = 14.7 μM, so that V max/ K m = 22 · 10 −2/min per unit as compared to 8 · 10 −2 for the commonly used substrate inosine. The permissible initial substrate concentration range is 5–100 μM. Enzyme activity may also be monitored spectrophotometrically. For 7-methylinosine, V max/ K m is much lower, 2.4 · 10 −2, but its 10-fold higher fluorescence partially compensates for this, and permits the use of initial substrate concentrations in the range 1–500 μM. At neutral pH both substrates are mixtures of cationic and zwitterionic forms. Measurements of pH-dependence of kinetic constants indicated that the cationic forms are the preferred substrates, whereas the monoanion of inosine appears to be almost as good a substrate as the neutral form. With 7-methylguanosine as substrate, and monitoring of activity fluorimetrically and spectrophotometrically, inhibition constants were measured for several known inhibitors, and the results compared with those obtained with inosine as substrate, and with results reported for the enzyme from other sources.