TABLE 2.
Kinetic parameters and NAD+ binding affinity of Scabin and Q158A/E160A variant
| Parametera | WT | Q158A/E160A |
|---|---|---|
| Km(GH) (μm) | 68 ± 3 | NDb |
| kcat(GH) (min−1) | 94 ± 2 | 0.31 ± 0.02 |
| KD(NAD+) (μm)c | 70 ± 3 | 86 ± 7 |
| K0.5(dG) (μm) | 302 ± 12 | ND |
| kcat(dG) (min−1) | 14 ± 1 | ND |
| kcat/Km(GH) (m−1 min−1) | 1.4 × 106 | ND |
| kcat/Km(dG) (m−1 min−1) | 4.6 × 104 | ND |
a Kinetic parameters were obtained as described under “Experimental Procedures” and represent the mean ± S.D. All results represent triplicate measurements for at least three separate experiments.
b ND, not determined.
c The KD value for the NAD+ substrate represents the binding affinity of NAD+ for the active site of Scabin and was measured by the Trp quenching caused by NAD+ docking within the active site of the enzyme (see “Experimental Procedures”). All results represent triplicate measurements for three separate experiments, mean ± S.D.