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Team:UC Davis/Protein Engineering MM
From 2014.igem.org
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 |
