Table I.
Kinetic characteristics for hPNP-Y88Xa
| Variant |
kcat (s−1) |
KM (µM) |
Kcat/KM (s−1 M−1) |
|||
|---|---|---|---|---|---|---|
| Inosine | ddI | Inosine | ddI | Inosine (×102) | ddI | |
| Wild type | 43.9 ± 0.6 | 0.9 ± 0.01 | 48 ± 2 | 1030 ± 20 | 9210 ± 140 | 875 ± 8 |
| Y88F | 28.3 ± 0.7 | 10.5 ± 0.1 | 73 ± 4 | 450 ± 10 | 3890 ± 110 | 23 140 ± 230 |
| Y88H | 8.6 ± 0.2 | 2.5 ± 0.1 | 74 ± 3 | 675 ± 20 | 1160 ± 23 | 3670 ± 60 |
| Y88W | 5.6 ± 0.1 | 0.09 ± 0.01 | 500 ± 25 | 980 ± 40 | 113 ± 3 | 91 ± 2 |
| Y88A | 17.6 ± 0.4 | 2.8 ± 0.05 | 80 ± 3 | 1670 ± 50 | 2190 ± 51 | 1670 ± 22 |
| Y88V | 2.4 ± 0.1 | 0.6 ± 0.01 | 1200 ± 60 | 4410 ± 160 | 19.6 ± 0.7 | 144 ± 3 |
| Y88L | 15.9 ± 0.3 | 6.1 ± 0.05 | 94 ± 4 | 391 ± 8 | 1690 ± 34 | 15 670 ± 130 |
| Y88I | 1.3 ± 0.1 | 0.5 ± 0.02 | 1420 ± 55 | 4670 ± 270 | 9.4 ± 0.3 | 115 ± 4 |
| Y88M | 12.8 ± 0.1 | 5.2 ± 0.1 | 345 ± 7 | 645 ± 20 | 371 ± 3 | 8090 ± 90 |
| Y88C | 5.5 ± 0.2 | 2.5 ± 0.1 | 170 ± 10 | 810 ± 30 | 321 ± 13 | 3070 ± 60 |
| Y88S | 8.8 ± 0.1 | 2.7 ± 0.1 | 430 ± 10 | 4060 ± 110 | 203 ± 3 | 652 ± 10 |
| Y88T | 2.3 ± 0.1 | 0.4 ± 0.01 | 1200 ± 70 | 8160 ± 280 | 19 ± 1 | 44 ± 1 |
| Y88N | 9.7 ± 0.2 | 1.5 ± 0.1 | 845 ± 25 | 9530 ± 540 | 114 ± 2 | 153 ± 6 |
| Y88Q | 1.4 ± 0.1 | 0.15 ± 0.01 | 855 ± 50 | 860 ± 25 | 17 ± 0.6 | 177 ± 2 |
| Y88D | 0.8 ± 0.1 | 0.02 ± 0.001 | 2600 ± 120 | 12 700 ± 500 | 2.9 ± 0.1 | 1.8 ± 0.5 |
| Y88E | 0.7 ± 0.1 | 0.01 ± 0.001 | 4860 ± 410 | 14 900 ± 1200 | 1.4 ± 0.1 | 0.9 ± 0.1 |
| Y88K | ND | ND | ND | ND | ND | ND |
| Y88R | 0.01 ± 0.001 | ND | 370 ± 20 | ND | 0.0184 ± 0.0005 | ND |
aAssay conditions: 50 mM phosphate buffer, pH 7, 50 mM HEPES, pH 7, 0.075% Triton X-100, 1 mM INT, xanthine oxidase. Inosine: 20–200 or 100–1000 µM. ddI: 250–2500 or 750–7500 µM. Enzymes were assayed at concentrations which ensure steady state conditions.