"
Team:UC Davis/Protein Engineering MM
From 2014.igem.org
(Difference between revisions)
| Line 50: | Line 50: | ||
<tr> | <tr> | ||
| - | <th colspan=" | + | <th colspan="9", text-align="center">Kinetic Constants of WT <i>Escherichia coli</i> Aldehyde Dehydrogenase</th> |
</tr> | </tr> | ||
| - | <tr>< | + | <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>Propionaldehyde</td><td>8 | + | <tr><td>Propionaldehyde</td><td>8</td><td>0.27</td><td>782.87</td><td>50.83</td><td>N/A</td><td>N/A</td><td>10221.04</td><td>748</td></tr> |
| - | <tr><td>Butyraldehyde</td><td>6.27</td><td>0.28</td><td>196.68</td><td>27.65</td><td>31867.74</td><td>4700.95</td></tr> | + | <tr><td>Butyraldehyde</td><td>6.27</td><td>0.28</td><td>196.68</td><td>27.65</td><td>N/A</td><td>N/A</td><td>31867.74</td><td>4700.95</td></tr> |
| - | <tr><td>Pentanal</td><td>6.18</td><td>0.22</td><td>45.92</td><td>6.77</td><td>134598 | + | <tr><td>Pentanal</td><td>6.18</td><td>0.22</td><td>45.92</td><td>6.77</td><td>N/A</td><td>N/A</td><td>134598</td><td>20398.49</td></tr> |
| - | <tr><td>Hexanal</td><td> | + | <tr><td>Hexanal</td><td>9.021</td><td>2.143</td><td>35.754</td><td>19.889</td><td>754.199</td><td>416.623</td><td>252307.4341</td><td>152614.3748</td></tr> |
| - | <tr><td>Heptanal</td><td | + | <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>64580</td><td>3940</td></tr> |
| - | <tr><td>Octanal</td><td>4.53</td><td>0.61</td><td>40.33</td><td>23.41</td><td>112265.52</td><td>66927.82</td></tr> | + | <tr><td>Octanal</td><td>4.53</td><td>0.61</td><td>40.33</td><td>23.41</td><td>N/A</td><td>N/A</td><td>112265.52</td><td>66927.82</td></tr> |
| - | <tr><td>Nonanal</td><td>3.99</td><td>0.44</td><td>27.62</td><td>14. | + | <tr><td>Nonanal</td><td>3.99</td><td>0.44</td><td>27.62</td><td>14.1</td><td>N/A</td><td>N/A</td><td>144584.42</td><td>75534.7</td></tr> |
| - | <tr><td>Decanal</td><td | + | <tr><td>Decanal</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>50120</td><td>3230</td></tr> |
| - | <tr><td>E-2-Pentenal</td><td>0.94</td><td>0.05</td><td>99.33</td><td>20.74</td><td>9480.51</td><td>2054.66</td></tr> | + | <tr><td>E-2-Pentenal</td><td>0.94</td><td>0.05</td><td>99.33</td><td>20.74</td><td>N/A</td><td>N/A</td><td>9480.51</td><td>2054.66</td></tr> |
| - | <tr><td>E-2-Hexenal</td><td>1.29</td><td>0.04</td><td>31.16</td><td>4.84</td><td>41478.41</td><td>6599.45</td></tr> | + | <tr><td>E-2-Hexenal</td><td>1.29</td><td>0.04</td><td>31.16</td><td>4.84</td><td>N/A</td><td>N/A</td><td>41478.41</td><td>6599.45</td></tr> |
| - | <tr><td>E-2-Octenal</td><td>0.99</td><td>0.03</td><td>12.27</td><td>1.86</td><td>81100.47</td><td>12529.78</td></tr> | + | <tr><td>E-2-Octenal</td><td>0.99</td><td>0.03</td><td>12.27</td><td>1.86</td><td>N/A</td><td>N/A</td><td>81100.47</td><td>12529.78</td></tr> |
| - | <tr><td>E-2-Nonenal</td><td>0.87</td><td>0.02</td><td>9. | + | <tr><td>E-2-Nonenal</td><td>0.87</td><td>0.02</td><td>9.6</td><td>1.16</td><td>N/A</td><td>N/A</td><td>90621.93</td><td>11127.45</td></tr> |
| - | <tr><td>E-2-Decenal</td><td>0.66</td><td>0.01</td><td>13.34</td><td>1.37</td><td>49715.95</td><td>5217.05</td></tr> | + | <tr><td>E-2-Decenal</td><td>0.66</td><td>0.01</td><td>13.34</td><td>1.37</td><td>N/A</td><td>N/A</td><td>49715.95</td><td>5217.05</td></tr> |
| - | <tr><td>E,E-2,4-Heptadienal</td><td>0.68</td><td>0.11</td><td>142.08</td><td>75.99</td><td>4809.31</td><td>2682.89</td></tr> | + | <tr><td>E,E-2,4-Heptadienal</td><td>0.68</td><td>0.11</td><td>142.08</td><td>75.99</td><td>N/A</td><td>N/A</td><td>4809.31</td><td>2682.89</td></tr> |
| - | <tr><td>E,E-2,4-Nonadienal</td><td | + | <tr><td>E,E-2,4-Nonadienal</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>1569</td><td>221.9</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>< | + | <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> |
| - | < | + | |
</table> | </table> | ||
| + | |||
<table id="t02"> | <table id="t02"> | ||
Revision as of 08:40, 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 | kcat Error | KM | KM Error | kcat/KM | kcat/KM Error |
| Propionaldehyde | 7.57 | 1.24 | 2575.71 | 559.82 | 2937.45 | 800.31 |
| Butyraldehyde | 4.42 | 0.12 | 532.70 | 32.00 | 8306.37 | 546.34 |
| Pentanal | 5.44 | 0.35 | 165.35 | 35.25 | 32877.93 | 7328.72 |
| Hexanal | 7.29 | 0.37 | 38.93 | 8.48 | 187159.14 | 41828.31 |
| Heptanal | 5.87 | 0.38 | 26.17 | 7.87 | 224363.85 | 68987.72 |
| Octanal | 6.55 | 0.45 | 84.25 | 21.41 | 77758.66 | 20464.40 |
| Nonanal | 6.41 | 0.31 | 75.29 | 13.54 | 85145.04 | 15837.70 |
| Decanal | 4.79 | 0.18 | 47.93 | 7.32 | 99954.31 | 15711.24 |
| E-2-Pentenal | ND | ND | ND | ND | ND | ND |
| E-2-Hexenal | 0.22 | 0.02 | 28.11 | 9.85 | 7797.24 | 2796.55 |
| E-2-Octenal | Unable to fit data to Michaelis-Menten | |||||
| E-2-Nonenal | Unable to fit data to Michaelis-Menten | |||||
| E-2-Decenal | Unable to fit data to Michaelis-Menten | |||||
| E,E-2,4-Heptadienal | ND | ND | ND | ND | ND | ND |
| E,E-2,4-Nonadienal | ND | ND | ND | ND | ND | ND |
| E,E-2,4-Decadienal | ND | ND | ND | ND | ND | ND |
| Kinetic Constants of WT Rattus norvegicus (Rat) Aldehyde Dehydrogenase | ||||||
|---|---|---|---|---|---|---|
| Substrate | kcat | kcat Error | KM | KM Error | kcat/KM | kcat/KM Error |
| Propionaldehyde | 19.90 | 0.51 | 1002.36 | 45.02 | 19856.17 | 1026.79 |
| Butyraldehyde | 12.46 | 0.22 | 261.02 | 13.60 | 47740.02 | 2629.46 |
| Pentanal | 10.51 | 0.44 | 118.05 | 17.28 | 89060.79 | 13565.38 |
| Hexanal | 8.28 | 0.66 | 32.90 | 11.62 | 251744.09 | 91148.58 |
| Heptanal | Unable to fit data to Michaelis-Menten | |||||
| Octanal | 6.62 | 0.57 | 107.94 | 32.67 | 61315.18 | 19282.67 |
| Nonanal | 7.62 | 0.68 | 88.69 | 29.02 | 85930.06 | 29145.22 |
| Decanal | 9.06 | 0.66 | 93.84 | 24.92 | 96541.80 | 26598.58 |
| E-2-Pentenal | 8.68 | 0.54 | 694.05 | 86.75 | 12509.32 | 1745.41 |
| E-2-Hexenal | 8.54 | 0.15 | 220.14 | 11.54 | 38785.27 | 2139.18 |
| E-2-Octenal | 7.51 | 0.47 | 65.81 | 16.08 | 114129.56 | 28807.70 |
| E-2-Nonenal | 9.50 | 0.58 | 75.41 | 17.52 | 125987.94 | 30284.84 |
| E-2-Decenal | 6.05 | 0.25 | 58.69 | 9.63 | 103066.45 | 17434.71 |
| E,E-2,4-Heptadienal | 6.01 | 0.31 | 31.77 | 7.40 | 189320.39 | 45208.07 |
| E,E-2,4-Nonadienal | 6.44 | 0.47 | 49.38 | 14.83 | 130374.03 | 40304.11 |
| E,E-2,4-Decadienal | 6.32 | 0.33 | 30.75 | 7.27 | 205360.25 | 49735.07 |
