Team:UC Davis/Protein Engineering MM

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UC Davis iGEM 2014

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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.

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	532.7	32	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	591.1	152.7	123500.8425	33883.89213
Octanal	16.214	5.973	301.64	146.694	547.52	335.184	53752.81793	32794.34307
Nonanal	10.45	1.878	161.828	45.71	1292.604	584.893	64574.73367	21618.61449
Decanal	6.8794	0.7062	91.5363	17.3332	1764.7464	570.9936	75154.88391	16187.92314
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	2056.7	237.6
E-2-Nonenal	N/A	N/A	N/A	N/A	N/A	N/A	1554.5	261.5
E-2-Decenal	N/A	N/A	N/A	N/A	N/A	N/A	1299.1	210.5
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	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

Team:UC Davis/Protein Engineering MM

From 2014.igem.org

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Experimentally Derived Kinetic Constants for Our Aldehyde Dehydrogenases