Team:UC Davis/Protein Engineering MM

From 2014.igem.org

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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.<br><br>
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.<br><br>
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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))].
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Revision as of 09:02, 17 October 2014

UC Davis iGEM 2014

Design

Design

Build

Build

Test

Test

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))].
Kinetic Constants of WT Escherichia coli Aldehyde Dehydrogenase
Substratekcat (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)
Propionaldehyde80.27782.8750.83N/AN/A10221.04748
Butyraldehyde6.270.28196.6827.65N/AN/A31867.744700.95
Pentanal6.180.2245.926.77N/AN/A13459820398.49
Hexanal9.0212.14335.75419.889754.199416.623252307.4341152614.3748
HeptanalN/AN/AN/AN/AN/AN/A645803940
Octanal4.530.6140.3323.41N/AN/A112265.5266927.82
Nonanal3.990.4427.6214.1N/AN/A144584.4275534.7
DecanalN/AN/AN/AN/AN/AN/A501203230
E-2-Pentenal0.940.0599.3320.74N/AN/A9480.512054.66
E-2-Hexenal1.290.0431.164.84N/AN/A41478.416599.45
E-2-Octenal0.990.0312.271.86N/AN/A81100.4712529.78
E-2-Nonenal0.870.029.61.16N/AN/A90621.9311127.45
E-2-Decenal0.660.0113.341.37N/AN/A49715.955217.05
E,E-2,4-Heptadienal0.680.11142.0875.99N/AN/A4809.312682.89
E,E-2,4-NonadienalN/AN/AN/AN/AN/AN/A1569221.9
E,E-2,4-DecadienalNDNDNDNDNDNDNDND
Kinetic Constants of W176V Escherichia coli Aldehyde Dehydrogenase
Substratekcat (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)
Propionaldehyde7.571.242575.71559.82N/AN/A2937.45800.31
Butyraldehyde4.420.12532.7032.00N/AN/A8306.37546.34
Pentanal5.440.35165.3535.25N/AN/A32877.937328.72
Hexanal7.290.3738.938.48N/AN/A187159.1441828.31
Heptanal12.461.76100.8923.73591.10152.70123500.8433883.89
Octanal16.215.97301.64146.69547.52335.1853752.8232794.34
Nonanal10.451.88161.8345.711292.60584.8964574.7321618.61
Decanal6.880.7191.5417.331764.75570.9975154.8816187.92
E-2-PentenalNDNDNDNDNDNDNDND
E-2-Hexenal0.220.0228.119.85N/AN/A7797.242796.55
E-2-OctenalN/AN/AN/AN/AN/AN/A2056.70237.60
E-2-NonenalN/AN/AN/AN/AN/AN/A1554.50261.50
E-2-DecenalN/AN/AN/AN/AN/AN/A1299.10210.50
E,E-2,4-HeptadienalNDNDNDNDNDNDNDND
E,E-2,4-NonadienalNDNDNDNDNDNDNDND
E,E-2,4-DecadienalNDNDNDNDNDNDNDND
Kinetic Constants of WT Rattus norvegicus (Rat) Aldehyde Dehydrogenase
Substratekcat (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)
Substratekcat (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)
Propionaldehyde19.900.511002.3645.02N/AN/A19856.171026.79
Butyraldehyde12.460.22261.0213.60N/AN/A47740.022629.46
Pentanal10.510.44118.0517.28N/AN/A89060.7913565.38
Hexanal8.280.6632.9011.62N/AN/A251744.0991148.58
HeptanalN/AN/AN/AN/AN/AN/A85934.007269.00
Octanal6.620.57107.9432.67N/AN/A61315.1819282.67
Nonanal7.620.6888.6929.02N/AN/A85930.0629145.22
Decanal9.060.6693.8424.92N/AN/A96541.8026598.58
E-2-Pentenal8.680.54694.0586.75N/AN/A12509.321745.41
E-2-Hexenal8.540.15220.1411.54N/AN/A38785.272139.18
E-2-Octenal7.510.4765.8116.08N/AN/A114129.5628807.70
E-2-Nonenal9.500.5875.4117.52N/AN/A125987.9430284.84
E-2-Decenal6.050.2558.699.63N/AN/A103066.4517434.71
E,E-2,4-Heptadienal6.010.3131.777.40N/AN/A189320.3945208.07
E,E-2,4-Nonadienal6.440.4749.3814.83N/AN/A130374.0340304.11
E,E-2,4-Decadienal6.320.3330.757.27N/AN/A205360.2549735.07

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