Table 4.
Thermal stability and ligand binding analysis of the wild-type and mutant dehydroquinate synthase enzymes
| Sample | Ligand | Tm (°C) | ΔH × 10−5 (kcal mole−1) | Error +/−×10−3 | Δ-5HVH × 10 (kcal mole−1) | KD (μM) |
| WT | Nonea | 42.77 (0.07) | 1.30 (0.03) | 3.2 | 1.27 (0.04) | |
| Zinca | 50.76 (0.09) | 1.39 (0.04) | 4.4 | 1.34 (0.05) | 0.15 | |
| Zincb | 50.55 (0.12) | 1.29 (0.05) | 5.1 | 1.27 (0.06) | ||
| DAHP | 52.50 (0.08) | 1.36 (0.04) | 3.6 | 1.33 (0.04) | 440 | |
| NAD+ | 54.62 (0.05) | 1.55 (0.03) | 3.0 | 1.51 (0.04) | 180 | |
| H275L | Nonea | 41.49 (0.09) | 1.21 (0.04) | 3.8 | 1.20 (0.05) | |
| Zinca | 52.19 (0.13) | 1.21 (0.05) | 4.6 | 1.10 (0.05) | 0.45 | |
| Zincb | 51.99 (0.22) | 1.19 (0.09) | 8.7 | 1.20 (0.11) | ||
| DAHP | 53.79 (0.17) | 1.23 (0.07) | 6.6 | 1.20 (0.08) | 320 | |
| NAD+ | 55.60 (0.07) | 1.30 (0.03) | 2.9 | 1.26 (0.04) | 97 | |
| R264A | Nonea | 43.40 (0.08) | 1.35 (0.04) | 4.4 | 1.37 (0.05) | |
| Zinca | 51.59 (0.11) | 1.45 (0.06) | 5.9 | 1.45 (0.07) | 0.11 | |
| Zincb | 50.79 (0.07) | 1.51 (0.04) | 4.4 | 1.48 (0.05) | ||
| DAHP | 52.11 (0.10) | 1.52 (0.06) | 5.9 | 1.47 (0.08) | 630 | |
| NAD+ | 55.72 (0.10) | 1.69 (0.08) | 7.5 | 1.68 (0.09) | 30 | |
| K152A | Nonea | 41.90 (0.40) | 1.19 (0.04) | 3.7 | 1.23 (0.05) | |
| Zinca | 49.57 (0.11) | 1.34 (0.05) | 5.1 | 1.28 (0.06) | 0.28 | |
| Zincb | 49.28 (0.11) | 1.43 (0.05) | 5.2 | 1.34 (0.07) | ||
| DAHP | 50.58 (0.18) | 1.45 (0.10) | 9.6 | 1.42 (0.12) | 690 | |
| NAD+ | 53.20 (0.13) | 1.73 (0.10) | 11.0 | 1.80 (0.15) | 73 | |
| R130A | Nonea | 41.20 (0.09) | 1.07 (0.03) | 3.2 | 1.09 (0.04) | |
| Zinca | 50.78 (0.09) | 1.37 (0.03) | 2.8 | 1.32 (0.03) | 0.16 | |
| Zincb | 50.82 (0.09) | 1.22 (0.04) | 4.0 | 1.27 (0.05) | ||
| DAHP | 51.48 (0.17) | 1.30 (0.03) | 2.8 | 1.25 (0.09) | 2300 | |
| NAD+ | 53.71 (0.13) | 1.42 (0.07) | 6.6 | 1.34 (0.08) | 238 | |
| R130K | Nonea | 41.60 (0.09) | 1.05 (0.03) | 2.7 | 1.01 (0.03) | |
| Zinca | 50.73 (0.09) | 1.31 (0.04) | 4.0 | 1.23 (0.05) | 0.14 | |
| Zincb | 50.52 (0.05) | 1.27 (0.02) | 2.2 | 1.32 (0.03) | ||
| DAHP | 51.86 (0.06) | 1.33 (0.03) | 2.8 | 1.35 (0.04) | 820 | |
| NAD+ | 54.14 (0.05) | 1.43 (0.02) | 2.34 | 1.41 (0.03) | 130 |
a For zinc binding, samples were treated to remove all zinc then a specified amount of zinc was added to the apoenzyme.
b For substrate binding, samples were prepared in the presence of zinc and dialyzed against 25 μM zinc prior to use.
Midpoint transition temperatures (Tm), calorimetric (ΔH) and van’t Hoff (ΔHVH) enthalpies for thermal unfolding determined by DSC in the presence and absence of ligands. The KD values for the binding of Zn2+, NAD+, and DAHP to the wild-type and mutant DHQS enzymes are derived from Tm shifts. The measured enthalpy changes, ΔH, and the calculated ΔHVH values for each experiment are shown. For each measurement the standard error is given in parentheses. Although the unfolding transitions are irreversible under the conditions we report here, the ratio of the calorimetric to van’t Hoff enthalpies, ΔHcal/3HVH are close to unity consistent with cooperative unfolding of a monomeric protein unit.