Table 1 Kinetic parameters for AncCARs and their cofactors.

From: Highly thermostable carboxylic acid reductases generated by ancestral sequence reconstruction

 

ATP

NADPH

AncCAR-A

  

 kcat (min−1)

340 ± 2.7

386.4 ± 11.1

 KM (µM)

76.8 ± 4.3

54.8 ± 5.1

 kcat/KM (min−1 µM−1)

4.4 ± 0.3

7.1 ± 0.7

AncCAR-PA

  

 kcat (min−1)

392.3 ± 11.2

482.2 ± 15

 KM (µM)

69.1 ± 6.7

58.5 ± 5.0

 kcat/KM (min−1 µM−1)

5.7 ± 0.6

8.2 ± 0.8

AncCAR-PF

  

 kcat (min−1)

219.2 ± 3.1

230.6 ± 2.5

 KM (µM)

42.9 ± 2.3

29.0 ± 1.2

 kcat/KM (min−1 µM−1)

5.1 ± 0.3

8.0 ± 0.3

ExCARs10

  

 KM (μM)

64–84

24–36

  1. Rates of AncCAR activity with ATP and NADPH were determined using a 12 point, 1.7× dilution series of substrate, with concentrations starting at 800 μM. Each concentration was investigated using three experimental replicates for each concentration point. Data were fitted to the Michaelis–Menten equation using GraphPad v.7.0. Graphs are shown in Supplementary Fig. 12. Errors show SEM
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