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Team:UC Davis/Protein Engineering MM
From 2014.igem.org
(Difference between revisions)
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<p> | <p> | ||
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| - | + | <b>Stock Solutions (Unless stated, all solutions were prepared in 11.1mM Potassium Phosphate, 111.1mM Potassium Chloride)</b><br> | |
| - | + | ||
| - | <b>Stock Solutions (Unless stated, all solutions were prepared in 11.1mM Potassium Phosphate, 111.1mM Potassium Chloride | + | |
Base: 1.54% Tween 20 (v/v), 1.11mM Dithiothreitol (DTT)<br> | Base: 1.54% Tween 20 (v/v), 1.11mM Dithiothreitol (DTT)<br> | ||
Substrate: 10x final concentration of aldehyde in isopropyl alcohol<br> | Substrate: 10x final concentration of aldehyde in isopropyl alcohol<br> | ||
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Order of addition to initiate assay: (1) 130uL substrate, (2) 20uL aldehyde, (3) 50uL enzyme. Assays were conducted in a Costar 96 well flat bottom plate. The activities of all aldehyde dehydrogenases were measured by the accumulation of NADH resulting from the oxidation of the assayed substrates. All assays were performed by incubating 18.72nM aldehyde dehydrogenase enzyme with substrate in a 200µL reaction containing 10mM phosphate buffer containing 100mM potassium chloride (pH 7.4), 1mM NAD+, 1mM DTT, 1% Tween 20, 10% isoproyl alcohol, and substrate. All aldehyde dehydrogenases were assayed against substrate concentrations of 1000uM, 500uM, 100uM, 50uM, 10uM, and 5uM. The change in absorbance of NADH at a wavelength of 340nm was measured using Biotek Epoch and Synergy microplate spectrophotometers. Kinetic constants were derived from the steady-state velocity curves using R. Curves were fit linearly [V=(kcat/KM)S], Michaelis-Menten [V=Vmax*S/(Km+S)], or substrate inhibition [V=Vmax*S/(Km+S*(1+S/Ki))]. | Order of addition to initiate assay: (1) 130uL substrate, (2) 20uL aldehyde, (3) 50uL enzyme. Assays were conducted in a Costar 96 well flat bottom plate. The activities of all aldehyde dehydrogenases were measured by the accumulation of NADH resulting from the oxidation of the assayed substrates. All assays were performed by incubating 18.72nM aldehyde dehydrogenase enzyme with substrate in a 200µL reaction containing 10mM phosphate buffer containing 100mM potassium chloride (pH 7.4), 1mM NAD+, 1mM DTT, 1% Tween 20, 10% isoproyl alcohol, and substrate. All aldehyde dehydrogenases were assayed against substrate concentrations of 1000uM, 500uM, 100uM, 50uM, 10uM, and 5uM. The change in absorbance of NADH at a wavelength of 340nm was measured using Biotek Epoch and Synergy microplate spectrophotometers. Kinetic constants were derived from the steady-state velocity curves using R. Curves were fit linearly [V=(kcat/KM)S], Michaelis-Menten [V=Vmax*S/(Km+S)], or substrate inhibition [V=Vmax*S/(Km+S*(1+S/Ki))]. | ||
| - | + | <br><br> | |
| + | <b>Key:</b><br> | ||
| + | N/A: Not applicable<br> | ||
| + | ND: No detection of activity<br> | ||
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</tr> | </tr> | ||
| - | <tr><th>Substrate</th><th>kcat (s-1)</th><th>kcat Error | + | <tr><th>Substrate</th><th>kcat<br>(s-1)</th><th>kcat Error<br>(s-1)</th><th>KM<br>(uM)</th><th>KM Error<br>(uM)</th><th>Ki<br>(uM)</th><th>Ki Error<br>(uM)</th><th>kcat/KM<br>(M-1s-1)</th><th>kcat/KM Error<br>(M-1s-1)</th></tr> |
<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>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>N/A</td><td>N/A</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>N/A</td><td>N/A</td><td>134598</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>N/A</td><td>N/A</td><td>134598</td><td>20398.49</td></tr> | ||
| - | <tr><td>Hexanal</td><td>9. | + | <tr><td>Hexanal</td><td>9.02</td><td>2.14</td><td>35.75</td><td>19.88</td><td>754.20</td><td>416.62</td><td>252307.43</td><td>152614.37</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>64580</td><td>3940</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>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>N/A</td><td>N/A</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> | ||
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</table> | </table> | ||
| + | <p> | ||
| + | <a href="https://static.igem.org/mediawiki/2014/9/93/Ecoli_Linear.pdf" class="brightlink">Download and view the linear fits for WT <i>Escherichia coli</i> Aldehyde Dehydrogenase here</a><br> | ||
| + | </p> | ||
| + | <p> | ||
| + | <a href="https://static.igem.org/mediawiki/2014/6/66/Ecoli_MM.pdf" class="brightlink">Download and view the Michaelis-Menten fits for WT <i>Escherichia coli</i> Aldehyde Dehydrogenase here</a><br> | ||
| + | </p> | ||
| + | |||
| + | <p> | ||
| + | <a href="https://static.igem.org/mediawiki/2014/1/1d/Ecoli_SI.pdf" class="brightlink">Download and view the substrate inhibition fits for WT <i>Escherichia coli</i> Aldehyde Dehydrogenase here</a><br> | ||
| + | </p> | ||
<table id="t02"> | <table id="t02"> | ||
<tr> | <tr> | ||
| - | <th colspan="9", text-align="center">Kinetic Constants of | + | <th colspan="9", text-align="center">Kinetic Constants of W176Q <i>Escherichia coli</i> Aldehyde Dehydrogenase</th> |
</tr> | </tr> | ||
| - | <tr><th>Substrate</th><th>kcat (s-1)</th><th>kcat Error | + | <tr><th>Substrate</th><th>kcat<br>(s-1)</th><th>kcat Error<br>(s-1)</th><th>KM<br>(uM)</th><th>KM Error<br>(uM)</th><th>Ki<br>(uM)</th><th>Ki Error<br>(uM)</th><th>kcat/KM<br>(M-1s-1)</th><th>kcat/KM Error<br>(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>532.70</td><td>32.00</td><td>N/A</td><td>N/A</td><td>8306.37</td><td>546.34</td></tr> | <tr><td>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> | ||
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<tr><td></td></tr> | <tr><td></td></tr> | ||
</table> | </table> | ||
| + | |||
| + | <p> | ||
| + | <a href="https://static.igem.org/mediawiki/2014/4/4f/W176Q_Linear.pdf" class="brightlink">Download and view the linear fits for W176Q <i>Escherichia coli</i> Aldehyde Dehydrogenase here</a><br> | ||
| + | </p> | ||
| + | |||
| + | <p> | ||
| + | <a href="https://static.igem.org/mediawiki/2014/5/5d/W176Q_MM.pdf" class="brightlink">Download and view the Michaelis-Menten fits for W176Q <i>Escherichia coli</i> Aldehyde Dehydrogenase here</a><br> | ||
| + | </p> | ||
| + | |||
| + | <p> | ||
| + | <a href="https://static.igem.org/mediawiki/2014/2/27/W176Q_SI.pdf" class="brightlink">Download and view the substrate inhibition fits for W176Q <i>Escherichia coli</i> Aldehyde Dehydrogenase here</a><br> | ||
| + | </p> | ||
<table id="t02"> | <table id="t02"> | ||
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</tr> | </tr> | ||
| - | <tr><th>Substrate</th><th>kcat (s-1)</th><th>kcat Error | + | <tr><th>Substrate</th><th>kcat<br>(s-1)</th><th>kcat Error<br>(s-1)</th><th>KM<br>(uM)</th><th>KM Error<br>(uM)</th><th>Ki<br>(uM)</th><th>Ki Error<br>(uM)</th><th>kcat/KM<br>(M-1s-1)</th><th>kcat/KM Error<br>(M-1s-1)</th></tr> |
| - | + | ||
<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>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>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> | ||
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</table> | </table> | ||
| + | <p> | ||
| + | <a href="https://static.igem.org/mediawiki/2014/4/43/Rat_Linear.pdf" class="brightlink">Download and view the linear fits for <i>Rattus norvegicus</i> (Rat) Aldehyde Dehydrogenase here</a><br> | ||
| + | </p> | ||
| + | |||
| + | <p> | ||
| + | <a href="https://static.igem.org/mediawiki/2014/4/49/Rat_MM.pdf" class="brightlink">Download and view the Michaelis-Menten fits for <i>Rattus norvegicus</i> (Rat) Aldehyde Dehydrogenase here</a><br> | ||
| + | </p> | ||
</div> | </div> | ||
Latest revision as of 00:08, 18 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.
Stock Solutions (Unless stated, all solutions were prepared in 11.1mM Potassium Phosphate, 111.1mM Potassium Chloride)
Base: 1.54% Tween 20 (v/v), 1.11mM Dithiothreitol (DTT)
Substrate: 10x final concentration of aldehyde in isopropyl alcohol
Enzyme: 0.004mg/mL Aldehyde Dehydrogenase enzyme, 4mM NAD+, 1.11mM Dithiothreitol (DTT)
Order of addition to initiate assay: (1) 130uL substrate, (2) 20uL aldehyde, (3) 50uL enzyme. Assays were conducted in a Costar 96 well flat bottom plate. The activities of all aldehyde dehydrogenases were measured by the accumulation of NADH resulting from the oxidation of the assayed substrates. All assays were performed by incubating 18.72nM aldehyde dehydrogenase enzyme with substrate in a 200µL reaction containing 10mM phosphate buffer containing 100mM potassium chloride (pH 7.4), 1mM NAD+, 1mM DTT, 1% Tween 20, 10% isoproyl alcohol, and substrate. All aldehyde dehydrogenases were assayed against substrate concentrations of 1000uM, 500uM, 100uM, 50uM, 10uM, and 5uM. The change in absorbance of NADH at a wavelength of 340nm was measured using Biotek Epoch and Synergy microplate spectrophotometers. Kinetic constants were derived from the steady-state velocity curves using R. Curves were fit linearly [V=(kcat/KM)S], Michaelis-Menten [V=Vmax*S/(Km+S)], or substrate inhibition [V=Vmax*S/(Km+S*(1+S/Ki))].
Key:
N/A: Not applicable
ND: No detection of activity
| 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 | 9.02 | 2.14 | 35.75 | 19.88 | 754.20 | 416.62 | 252307.43 | 152614.37 |
| 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 |
Download and view the linear fits for WT Escherichia coli Aldehyde Dehydrogenase here
Download and view the Michaelis-Menten fits for WT Escherichia coli Aldehyde Dehydrogenase here
Download and view the substrate inhibition fits for WT Escherichia coli Aldehyde Dehydrogenase here
| Kinetic Constants of W176Q 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 |
Download and view the linear fits for W176Q Escherichia coli Aldehyde Dehydrogenase here
Download and view the Michaelis-Menten fits for W176Q Escherichia coli Aldehyde Dehydrogenase here
| 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 | 19.90 | 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 |
Download and view the linear fits for Rattus norvegicus (Rat) Aldehyde Dehydrogenase here
Download and view the Michaelis-Menten fits for Rattus norvegicus (Rat) Aldehyde Dehydrogenase here
