Table 1.
Kinetic parameters for enzymatic ribosylation of selected 8-azapurines in 25 mM HEPES buffer, pH 6.6, by α-d-ribose-1-phosphate, using various forms of PNP (nd = not determined). Standard errors are estimated to be ~15%.
| Substrate/Enzyme | Km (µM) | Vmax (Relative) * | Approximate Product Ratio: N9-riboside: N8-riboside:N7-riboside |
|---|---|---|---|
| 8-azaGua/calf PNP-wt | ~90 | 21 | 1:0:0 |
| 8-azaGua/calf PNP-N243D | >100 | >0.2 *** | 1:0:2 |
| 8-azaGua/E. coli PNP-wt | >200 | ~1 *** | 20:1:0 |
| 8-azaGua/E. coli PNP-D204N | nd | traces | predominantly N9 |
| 8-azaDaPur/calf PNP-wt | 60 | ~1 | 0:1:1 ** |
| 8-azaDaPur/calf PNP-N243D | 35 | 0.6 | 1:0:3 |
| 8-azaDaPur/E. coli PNP-wt | >200 | ~3 *** | 1:2:0 |
| 8-azaDaPur/E. coli PNP-D204N | >200 | ~0.4 *** | 10:1:0 |
* relative to guanine ribosylation under the same conditions (=100); ** this ratio is dependent on the reaction progress; *** reaction rates measured at 200 µM substrate.