"
Team:UC Davis/Protein Engineering MM
From 2014.igem.org
(Difference between revisions)
| Line 81: | Line 81: | ||
<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>7.57</td><td>1.24</td><td>2575.71</td><td>559.82</td><td>N/A</td><td>N/A</td><td>2937.45</td><td>800.31</td></tr> | <tr><td>Propionaldehyde</td><td>7.57</td><td>1.24</td><td>2575.71</td><td>559.82</td><td>N/A</td><td>N/A</td><td>2937.45</td><td>800.31</td></tr> | ||
| - | <tr><td>Butyraldehyde</td><td>4.42</td><td>0.12</td><td>532. | + | <tr><td>Butyraldehyde</td><td>4.42</td><td>0.12</td><td>532.70</td><td>32.00</td><td>N/A</td><td>N/A</td><td>8306.37</td><td>546.34</td></tr> |
<tr><td>Pentanal</td><td>5.44</td><td>0.35</td><td>165.35</td><td>35.25</td><td>N/A</td><td>N/A</td><td>32877.93</td><td>7328.72</td></tr> | <tr><td>Pentanal</td><td>5.44</td><td>0.35</td><td>165.35</td><td>35.25</td><td>N/A</td><td>N/A</td><td>32877.93</td><td>7328.72</td></tr> | ||
<tr><td>Hexanal</td><td>7.29</td><td>0.37</td><td>38.93</td><td>8.48</td><td>N/A</td><td>N/A</td><td>187159.14</td><td>41828.31</td></tr> | <tr><td>Hexanal</td><td>7.29</td><td>0.37</td><td>38.93</td><td>8.48</td><td>N/A</td><td>N/A</td><td>187159.14</td><td>41828.31</td></tr> | ||
| - | <tr><td>Heptanal</td><td>12.46</td><td>1.76</td><td>100.89</td><td>23.73</td><td>591. | + | <tr><td>Heptanal</td><td>12.46</td><td>1.76</td><td>100.89</td><td>23.73</td><td>591.10</td><td>152.70</td><td>123500.84</td><td>33883.89</td></tr> |
| - | <tr><td>Octanal</td><td>16. | + | <tr><td>Octanal</td><td>16.21</td><td>5.97</td><td>301.64</td><td>146.69</td><td>547.52</td><td>335.18</td><td>53752.82</td><td>32794.34</td></tr> |
| - | <tr><td>Nonanal</td><td>10.45</td><td>1. | + | <tr><td>Nonanal</td><td>10.45</td><td>1.88</td><td>161.83</td><td>45.71</td><td>1292.60</td><td>584.89</td><td>64574.73</td><td>21618.61</td></tr> |
| - | <tr><td>Decanal</td><td>6. | + | <tr><td>Decanal</td><td>6.88</td><td>0.71</td><td>91.54</td><td>17.33</td><td>1764.75</td><td>570.99</td><td>75154.88</td><td>16187.92</td></tr> |
<tr><td>E-2-Pentenal</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td></tr> | <tr><td>E-2-Pentenal</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td></tr> | ||
<tr><td>E-2-Hexenal</td><td>0.22</td><td>0.02</td><td>28.11</td><td>9.85</td><td>N/A</td><td>N/A</td><td>7797.24</td><td>2796.55</td></tr> | <tr><td>E-2-Hexenal</td><td>0.22</td><td>0.02</td><td>28.11</td><td>9.85</td><td>N/A</td><td>N/A</td><td>7797.24</td><td>2796.55</td></tr> | ||
| - | <tr><td>E-2-Octenal</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>2056. | + | <tr><td>E-2-Octenal</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>2056.70</td><td>237.60</td></tr> |
| - | <tr><td>E-2-Nonenal</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>1554. | + | <tr><td>E-2-Nonenal</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>1554.50</td><td>261.50</td></tr> |
| - | <tr><td>E-2-Decenal</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>1299. | + | <tr><td>E-2-Decenal</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>1299.10</td><td>210.50</td></tr> |
<tr><td>E,E-2,4-Heptadienal</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td></tr> | <tr><td>E,E-2,4-Heptadienal</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td></tr> | ||
<tr><td>E,E-2,4-Nonadienal</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td></tr> | <tr><td>E,E-2,4-Nonadienal</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td></tr> | ||
<tr><td>E,E-2,4-Decadienal</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td></tr> | <tr><td>E,E-2,4-Decadienal</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td></tr> | ||
| + | <tr><td></td></tr> | ||
</table> | </table> | ||
Revision as of 08:46, 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) |
| Propionaldehyde | 19.9 | 0.51 | 1002.36 | 45.02 | N/A | N/A | 19856.17 | 1026.79 |
| Butyraldehyde | 12.46 | 0.22 | 261.02 | 13.6 | 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 | 32.9 | 11.62 | N/A | N/A | 251744.09 | 91148.58 |
| Heptanal | N/A | N/A | N/A | N/A | N/A | N/A | 85934 | 7269 |
| 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 | 96541.8 | 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 | 28807.7 |
| E-2-Nonenal | 9.5 | 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 | 7.4 | 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 |
