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Team:UC Davis/Protein Engineering MM
From 2014.igem.org
(Difference between revisions)
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<tr><th>Substrate</th><th>kcat (s-1)</th><th>kcat Error (s-1)</th><th>KM (uM)</th><th>KM Error (uM)</th><th>Ki (uM)</th><th>Ki Error (uM)</th><th>kcat/KM (M-1s-1)</th><th>kcat/KM Error (M-1s-1)</th></tr> | <tr><th>Substrate</th><th>kcat (s-1)</th><th>kcat Error (s-1)</th><th>KM (uM)</th><th>KM Error (uM)</th><th>Ki (uM)</th><th>Ki Error (uM)</th><th>kcat/KM (M-1s-1)</th><th>kcat/KM Error (M-1s-1)</th></tr> | ||
| - | <tr><td>Propionaldehyde</td><td>19. | + | <tr><td>Substrate</td><td>kcat (s-1)</td><td>kcat Error (s-1)</td><td>KM (uM)</td><td>KM Error (uM)</td><td>Ki (uM)</td><td>Ki Error (uM)</td><td>kcat/KM (M-1s-1)</td><td>kcat/KM Error (M-1s-1)</td></tr> |
| - | <tr><td>Butyraldehyde</td><td>12.46</td><td>0.22</td><td>261.02</td><td>13. | + | <tr><td>Propionaldehyde</td><td>19.90</td><td>0.51</td><td>1002.36</td><td>45.02</td><td>N/A</td><td>N/A</td><td>19856.17</td><td>1026.79</td></tr> |
| + | <tr><td>Butyraldehyde</td><td>12.46</td><td>0.22</td><td>261.02</td><td>13.60</td><td>N/A</td><td>N/A</td><td>47740.02</td><td>2629.46</td></tr> | ||
<tr><td>Pentanal</td><td>10.51</td><td>0.44</td><td>118.05</td><td>17.28</td><td>N/A</td><td>N/A</td><td>89060.79</td><td>13565.38</td></tr> | <tr><td>Pentanal</td><td>10.51</td><td>0.44</td><td>118.05</td><td>17.28</td><td>N/A</td><td>N/A</td><td>89060.79</td><td>13565.38</td></tr> | ||
| - | <tr><td>Hexanal</td><td>8.28</td><td>0.66</td><td>32. | + | <tr><td>Hexanal</td><td>8.28</td><td>0.66</td><td>32.90</td><td>11.62</td><td>N/A</td><td>N/A</td><td>251744.09</td><td>91148.58</td></tr> |
| - | <tr><td>Heptanal</td><td>N/A</td><td>N/A</td><td>N/A</td><td>N/A</td><td>N/A</td><td>N/A</td><td>85934</td><td>7269</td></tr> | + | <tr><td>Heptanal</td><td>N/A</td><td>N/A</td><td>N/A</td><td>N/A</td><td>N/A</td><td>N/A</td><td>85934.00</td><td>7269.00</td></tr> |
<tr><td>Octanal</td><td>6.62</td><td>0.57</td><td>107.94</td><td>32.67</td><td>N/A</td><td>N/A</td><td>61315.18</td><td>19282.67</td></tr> | <tr><td>Octanal</td><td>6.62</td><td>0.57</td><td>107.94</td><td>32.67</td><td>N/A</td><td>N/A</td><td>61315.18</td><td>19282.67</td></tr> | ||
<tr><td>Nonanal</td><td>7.62</td><td>0.68</td><td>88.69</td><td>29.02</td><td>N/A</td><td>N/A</td><td>85930.06</td><td>29145.22</td></tr> | <tr><td>Nonanal</td><td>7.62</td><td>0.68</td><td>88.69</td><td>29.02</td><td>N/A</td><td>N/A</td><td>85930.06</td><td>29145.22</td></tr> | ||
| - | <tr><td>Decanal</td><td>9.06</td><td>0.66</td><td>93.84</td><td>24.92</td><td>N/A</td><td>N/A</td><td>96541. | + | <tr><td>Decanal</td><td>9.06</td><td>0.66</td><td>93.84</td><td>24.92</td><td>N/A</td><td>N/A</td><td>96541.80</td><td>26598.58</td></tr> |
<tr><td>E-2-Pentenal</td><td>8.68</td><td>0.54</td><td>694.05</td><td>86.75</td><td>N/A</td><td>N/A</td><td>12509.32</td><td>1745.41</td></tr> | <tr><td>E-2-Pentenal</td><td>8.68</td><td>0.54</td><td>694.05</td><td>86.75</td><td>N/A</td><td>N/A</td><td>12509.32</td><td>1745.41</td></tr> | ||
<tr><td>E-2-Hexenal</td><td>8.54</td><td>0.15</td><td>220.14</td><td>11.54</td><td>N/A</td><td>N/A</td><td>38785.27</td><td>2139.18</td></tr> | <tr><td>E-2-Hexenal</td><td>8.54</td><td>0.15</td><td>220.14</td><td>11.54</td><td>N/A</td><td>N/A</td><td>38785.27</td><td>2139.18</td></tr> | ||
| - | <tr><td>E-2-Octenal</td><td>7.51</td><td>0.47</td><td>65.81</td><td>16.08</td><td>N/A</td><td>N/A</td><td>114129.56</td><td>28807. | + | <tr><td>E-2-Octenal</td><td>7.51</td><td>0.47</td><td>65.81</td><td>16.08</td><td>N/A</td><td>N/A</td><td>114129.56</td><td>28807.70</td></tr> |
| - | <tr><td>E-2-Nonenal</td><td>9. | + | <tr><td>E-2-Nonenal</td><td>9.50</td><td>0.58</td><td>75.41</td><td>17.52</td><td>N/A</td><td>N/A</td><td>125987.94</td><td>30284.84</td></tr> |
<tr><td>E-2-Decenal</td><td>6.05</td><td>0.25</td><td>58.69</td><td>9.63</td><td>N/A</td><td>N/A</td><td>103066.45</td><td>17434.71</td></tr> | <tr><td>E-2-Decenal</td><td>6.05</td><td>0.25</td><td>58.69</td><td>9.63</td><td>N/A</td><td>N/A</td><td>103066.45</td><td>17434.71</td></tr> | ||
| - | <tr><td>E,E-2,4-Heptadienal</td><td>6.01</td><td>0.31</td><td>31.77</td><td>7. | + | <tr><td>E,E-2,4-Heptadienal</td><td>6.01</td><td>0.31</td><td>31.77</td><td>7.40</td><td>N/A</td><td>N/A</td><td>189320.39</td><td>45208.07</td></tr> |
<tr><td>E,E-2,4-Nonadienal</td><td>6.44</td><td>0.47</td><td>49.38</td><td>14.83</td><td>N/A</td><td>N/A</td><td>130374.03</td><td>40304.11</td></tr> | <tr><td>E,E-2,4-Nonadienal</td><td>6.44</td><td>0.47</td><td>49.38</td><td>14.83</td><td>N/A</td><td>N/A</td><td>130374.03</td><td>40304.11</td></tr> | ||
<tr><td>E,E-2,4-Decadienal</td><td>6.32</td><td>0.33</td><td>30.75</td><td>7.27</td><td>N/A</td><td>N/A</td><td>205360.25</td><td>49735.07</td></tr> | <tr><td>E,E-2,4-Decadienal</td><td>6.32</td><td>0.33</td><td>30.75</td><td>7.27</td><td>N/A</td><td>N/A</td><td>205360.25</td><td>49735.07</td></tr> | ||
| - | |||
</table> | </table> | ||
| + | |||
</div> | </div> | ||
Revision as of 08:48, 17 October 2014
Experimentally Derived Kinetic Constants for Our Aldehyde Dehydrogenases
Now that we had identified three aldehyde dehydrogenases with unique specificity profiles, we proceeded to experimentally determine the kinetic constants (kcat and KM) of these enzymes on each substrate in our assay conditions.
| Kinetic Constants of WT Escherichia coli Aldehyde Dehydrogenase | ||||||||
|---|---|---|---|---|---|---|---|---|
| Substrate | kcat (s-1) | kcat Error (s-1) | KM (uM) | KM Error (uM) | Ki (uM) | Ki Error (uM) | kcat/KM (M-1s-1) | kcat/KM Error (M-1s-1) |
| Propionaldehyde | 8 | 0.27 | 782.87 | 50.83 | N/A | N/A | 10221.04 | 748 |
| Butyraldehyde | 6.27 | 0.28 | 196.68 | 27.65 | N/A | N/A | 31867.74 | 4700.95 |
| Pentanal | 6.18 | 0.22 | 45.92 | 6.77 | N/A | N/A | 134598 | 20398.49 |
| Hexanal | 9.021 | 2.143 | 35.754 | 19.889 | 754.199 | 416.623 | 252307.4341 | 152614.3748 |
| Heptanal | N/A | N/A | N/A | N/A | N/A | N/A | 64580 | 3940 |
| Octanal | 4.53 | 0.61 | 40.33 | 23.41 | N/A | N/A | 112265.52 | 66927.82 |
| Nonanal | 3.99 | 0.44 | 27.62 | 14.1 | N/A | N/A | 144584.42 | 75534.7 |
| Decanal | N/A | N/A | N/A | N/A | N/A | N/A | 50120 | 3230 |
| E-2-Pentenal | 0.94 | 0.05 | 99.33 | 20.74 | N/A | N/A | 9480.51 | 2054.66 |
| E-2-Hexenal | 1.29 | 0.04 | 31.16 | 4.84 | N/A | N/A | 41478.41 | 6599.45 |
| E-2-Octenal | 0.99 | 0.03 | 12.27 | 1.86 | N/A | N/A | 81100.47 | 12529.78 |
| E-2-Nonenal | 0.87 | 0.02 | 9.6 | 1.16 | N/A | N/A | 90621.93 | 11127.45 |
| E-2-Decenal | 0.66 | 0.01 | 13.34 | 1.37 | N/A | N/A | 49715.95 | 5217.05 |
| E,E-2,4-Heptadienal | 0.68 | 0.11 | 142.08 | 75.99 | N/A | N/A | 4809.31 | 2682.89 |
| E,E-2,4-Nonadienal | N/A | N/A | N/A | N/A | N/A | N/A | 1569 | 221.9 |
| E,E-2,4-Decadienal | ND | ND | ND | ND | ND | ND | ND | ND |
| Kinetic Constants of W176V Escherichia coli Aldehyde Dehydrogenase | ||||||||
|---|---|---|---|---|---|---|---|---|
| Substrate | kcat (s-1) | kcat Error (s-1) | KM (uM) | KM Error (uM) | Ki (uM) | Ki Error (uM) | kcat/KM (M-1s-1) | kcat/KM Error (M-1s-1) |
| Propionaldehyde | 7.57 | 1.24 | 2575.71 | 559.82 | N/A | N/A | 2937.45 | 800.31 |
| Butyraldehyde | 4.42 | 0.12 | 532.70 | 32.00 | N/A | N/A | 8306.37 | 546.34 |
| Pentanal | 5.44 | 0.35 | 165.35 | 35.25 | N/A | N/A | 32877.93 | 7328.72 |
| Hexanal | 7.29 | 0.37 | 38.93 | 8.48 | N/A | N/A | 187159.14 | 41828.31 |
| Heptanal | 12.46 | 1.76 | 100.89 | 23.73 | 591.10 | 152.70 | 123500.84 | 33883.89 |
| Octanal | 16.21 | 5.97 | 301.64 | 146.69 | 547.52 | 335.18 | 53752.82 | 32794.34 |
| Nonanal | 10.45 | 1.88 | 161.83 | 45.71 | 1292.60 | 584.89 | 64574.73 | 21618.61 |
| Decanal | 6.88 | 0.71 | 91.54 | 17.33 | 1764.75 | 570.99 | 75154.88 | 16187.92 |
| E-2-Pentenal | ND | ND | ND | ND | ND | ND | ND | ND |
| E-2-Hexenal | 0.22 | 0.02 | 28.11 | 9.85 | N/A | N/A | 7797.24 | 2796.55 |
| E-2-Octenal | N/A | N/A | N/A | N/A | N/A | N/A | 2056.70 | 237.60 |
| E-2-Nonenal | N/A | N/A | N/A | N/A | N/A | N/A | 1554.50 | 261.50 |
| E-2-Decenal | N/A | N/A | N/A | N/A | N/A | N/A | 1299.10 | 210.50 |
| E,E-2,4-Heptadienal | ND | ND | ND | ND | ND | ND | ND | ND |
| E,E-2,4-Nonadienal | ND | ND | ND | ND | ND | ND | ND | ND |
| E,E-2,4-Decadienal | ND | ND | ND | ND | ND | ND | ND | ND |
| Kinetic Constants of WT Rattus norvegicus (Rat) Aldehyde Dehydrogenase | ||||||||
|---|---|---|---|---|---|---|---|---|
| Substrate | kcat (s-1) | kcat Error (s-1) | KM (uM) | KM Error (uM) | Ki (uM) | Ki Error (uM) | kcat/KM (M-1s-1) | kcat/KM Error (M-1s-1) |
| Substrate | kcat (s-1) | kcat Error (s-1) | KM (uM) | KM Error (uM) | Ki (uM) | Ki Error (uM) | kcat/KM (M-1s-1) | kcat/KM Error (M-1s-1) |
| Propionaldehyde | 19.90 | 0.51 | 1002.36 | 45.02 | N/A | N/A | 19856.17 | 1026.79 |
| Butyraldehyde | 12.46 | 0.22 | 261.02 | 13.60 | N/A | N/A | 47740.02 | 2629.46 |
| Pentanal | 10.51 | 0.44 | 118.05 | 17.28 | N/A | N/A | 89060.79 | 13565.38 |
| Hexanal | 8.28 | 0.66 | 32.90 | 11.62 | N/A | N/A | 251744.09 | 91148.58 |
| Heptanal | N/A | N/A | N/A | N/A | N/A | N/A | 85934.00 | 7269.00 |
| Octanal | 6.62 | 0.57 | 107.94 | 32.67 | N/A | N/A | 61315.18 | 19282.67 |
| Nonanal | 7.62 | 0.68 | 88.69 | 29.02 | N/A | N/A | 85930.06 | 29145.22 |
| Decanal | 9.06 | 0.66 | 93.84 | 24.92 | N/A | N/A | 96541.80 | 26598.58 |
| E-2-Pentenal | 8.68 | 0.54 | 694.05 | 86.75 | N/A | N/A | 12509.32 | 1745.41 |
| E-2-Hexenal | 8.54 | 0.15 | 220.14 | 11.54 | N/A | N/A | 38785.27 | 2139.18 |
| E-2-Octenal | 7.51 | 0.47 | 65.81 | 16.08 | N/A | N/A | 114129.56 | 28807.70 |
| E-2-Nonenal | 9.50 | 0.58 | 75.41 | 17.52 | N/A | N/A | 125987.94 | 30284.84 |
| E-2-Decenal | 6.05 | 0.25 | 58.69 | 9.63 | N/A | N/A | 103066.45 | 17434.71 |
| E,E-2,4-Heptadienal | 6.01 | 0.31 | 31.77 | 7.40 | N/A | N/A | 189320.39 | 45208.07 |
| E,E-2,4-Nonadienal | 6.44 | 0.47 | 49.38 | 14.83 | N/A | N/A | 130374.03 | 40304.11 |
| E,E-2,4-Decadienal | 6.32 | 0.33 | 30.75 | 7.27 | N/A | N/A | 205360.25 | 49735.07 |
