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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.
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 1x PBS (pH 7.4), 1mM NAD+, 1mM DTT, 1% Tween 20, 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))].
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 1x PBS (pH 7.4), 1mM NAD+, 1mM DTT, 1% Tween 20, 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))].
| 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) |
| 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 |
