Table 2.
Kinetic parameters for the enzymatic ribosylation of nucleobase analogs investigated in this work, in 40 mM HEPES buffer, pH 7, by α-d-ribose-1-phosphate, using various forms of PNP (wt = wild type). Standard errors are estimated to be ~15%.
| Substrate/Enzyme | Km
(µM) |
Vmax
(relative) * |
Approximate Product Ratio: N9: N7-riboside: N6-riboside |
|---|---|---|---|
| 1,N2-ethenoguanine /calf PNP-wt** |
nd | <0.5 | predominantly N9 |
| 1,N2-ethenoguanine /calf PNP-mutantN243D |
nd | - | - |
| 1,N2-ethenoguanine /E.coli PNP-wt |
48 | 18 | predominantly N9 |
| 1,N2-ethenoguanine /E.coli PNP-mutantD204N |
nd | >15 | predominantly N9 |
|
N2,3-ethenoguanine /calf PNP-wt |
- | <0.1 | - |
|
N2,3-ethenoguanine /E.coli PNP-wt |
- | <0.1 | - |
|
N2,3-etheno-O6-methylguanine /calf PNP-wt |
- | <0.1 | - |
|
N2,3-etheno-O6-methylguanine /E.coli PNP-wt |
~6 | ~0.3 | Nd |
| 1,N6-ethenoisoguanine /calf PNP-wt |
~160 | 1.6 | 1:1:10 |
| 1,N6-ethenoisoguanine /calf PNP- mutantN243D |
nd | ~1 | predominantly N6 |
| 1,N6-ethenoisoguanine /E.coli PNP-wt |
98 | ~24 | 1:4:0.1 |
| 1,N6-ethenoisoguanine /E.coli PNP-mutantD204N |
nd | >5 | 5:5:0 |
* relative to Vmax of guanine ribosylation under the same conditions (=100); error ~10%; ** wt = wild type.