"
Team:UC Davis/Protein Engineering MM
From 2014.igem.org
(Difference between revisions)
| Line 58: | Line 58: | ||
<tr><td>Pentanal</td><td>6.18</td><td>0.22</td><td>45.92</td><td>6.77</td><td>134598.00</td><td>20398.49</td></tr> | <tr><td>Pentanal</td><td>6.18</td><td>0.22</td><td>45.92</td><td>6.77</td><td>134598.00</td><td>20398.49</td></tr> | ||
<tr><td>Hexanal</td><td>5.37</td><td>0.53</td><td>12.65</td><td>6.36</td><td>424348.82</td><td>217516.13</td></tr> | <tr><td>Hexanal</td><td>5.37</td><td>0.53</td><td>12.65</td><td>6.36</td><td>424348.82</td><td>217516.13</td></tr> | ||
| - | <tr><td>Heptanal</td><td | + | <tr><td>Heptanal</td><td colspan="6", text-align="center">Unable to fit data to Michaelis-Menten</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>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>14.10</td><td>144584.42</td><td>75534.70</td></tr> | <tr><td>Nonanal</td><td>3.99</td><td>0.44</td><td>27.62</td><td>14.10</td><td>144584.42</td><td>75534.70</td></tr> | ||
| - | <tr><td>Decanal</td><td | + | <tr><td>Decanal</td><td colspan="6", text-align="center">Unable to fit data to Michaelis-Menten</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>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>41478.41</td><td>6599.45</td></tr> | ||
| Line 68: | Line 68: | ||
<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>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>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 colspan="6", text-align="center">Unable to fit data to Michaelis-Menten</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> | <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> | ||
<tr><td></td></tr> | <tr><td></td></tr> | ||
| Line 90: | Line 90: | ||
<tr><td>E-2-Pentenal</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></tr> | ||
<tr><td>E-2-Hexenal</td><td>0.22</td><td>0.02</td><td>28.11</td><td>9.85</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>7797.24</td><td>2796.55</td></tr> | ||
| - | <tr><td>E-2-Octenal</td><td | + | <tr><td>E-2-Octenal</td><td colspan="6", text-align="center">Unable to fit data to Michaelis-Menten</td></tr> |
| - | <tr><td>E-2-Nonenal</td><td | + | <tr><td>E-2-Nonenal</td><td colspan="6", text-align="center">Unable to fit data to Michaelis-Menten</td></tr> |
| - | <tr><td>E-2-Decenal</td><td | + | <tr><td>E-2-Decenal</td><td colspan="6", text-align="center">Unable to fit data to Michaelis-Menten</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></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></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></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></tr> | ||
| Line 110: | Line 110: | ||
<tr><td>Pentanal</td><td>10.51</td><td>0.44</td><td>118.05</td><td>17.28</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>89060.79</td><td>13565.38</td></tr> | ||
<tr><td>Hexanal</td><td>8.28</td><td>0.66</td><td>32.90</td><td>11.62</td><td>251744.09</td><td>91148.58</td></tr> | <tr><td>Hexanal</td><td>8.28</td><td>0.66</td><td>32.90</td><td>11.62</td><td>251744.09</td><td>91148.58</td></tr> | ||
| - | <tr><td>Heptanal</td><td | + | <tr><td>Heptanal</td><td colspan="6", text-align="center">Unable to fit data to Michaelis-Menten</td></tr> |
<tr><td>Octanal</td><td>6.62</td><td>0.57</td><td>107.94</td><td>32.67</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>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>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>85930.06</td><td>29145.22</td></tr> | ||
Revision as of 01:04, 16 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 | kcat Error | KM | KM Error | kcat/KM | kcat/KM Error |
| Propionaldehyde | 8.00 | 0.27 | 782.87 | 50.83 | 10221.04 | 748.00 |
| Butyraldehyde | 6.27 | 0.28 | 196.68 | 27.65 | 31867.74 | 4700.95 |
| Pentanal | 6.18 | 0.22 | 45.92 | 6.77 | 134598.00 | 20398.49 |
| Hexanal | 5.37 | 0.53 | 12.65 | 6.36 | 424348.82 | 217516.13 |
| Heptanal | Unable to fit data to Michaelis-Menten | |||||
| Octanal | 4.53 | 0.61 | 40.33 | 23.41 | 112265.52 | 66927.82 |
| Nonanal | 3.99 | 0.44 | 27.62 | 14.10 | 144584.42 | 75534.70 |
| Decanal | Unable to fit data to Michaelis-Menten | |||||
| E-2-Pentenal | 0.94 | 0.05 | 99.33 | 20.74 | 9480.51 | 2054.66 |
| E-2-Hexenal | 1.29 | 0.04 | 31.16 | 4.84 | 41478.41 | 6599.45 |
| E-2-Octenal | 0.99 | 0.03 | 12.27 | 1.86 | 81100.47 | 12529.78 |
| E-2-Nonenal | 0.87 | 0.02 | 9.60 | 1.16 | 90621.93 | 11127.45 |
| E-2-Decenal | 0.66 | 0.01 | 13.34 | 1.37 | 49715.95 | 5217.05 |
| E,E-2,4-Heptadienal | 0.68 | 0.11 | 142.08 | 75.99 | 4809.31 | 2682.89 |
| E,E-2,4-Nonadienal | Unable to fit data to Michaelis-Menten | |||||
| E,E-2,4-Decadienal | 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 |
