Table 2. Kinetic parameters of AarA and active site mutant using psTatA‐Förster resonance energy transfer (FRET) substrate
ProteinK0.5 (μM)Vmax (μM min−1)kcat (min−1)kcat/K0.5 (min−1 μM−1)Hill coefficient
AarA wt3.9 ± 0.32.3 × 10−2 ± 0.1 × 10−20.17 ± 0.074.3 × 10−2 ± 0.5 × 10−21.8 ± 0.1
AarA S150A00000
  • The purified substrate (0.13–7 μM) was incubated with AarA or mutant protein (0.135 μM) at 37°C for 2 h, and the emission intensity of CyPet at 475 nm and YPet at 530 nm was recorded with the excitation wavelength of 414 nm. The initial velocities were calculated for each substrate concentration. K0.5 and Vmax were obtained from fitting the data to the Hill's equation by plotting the initial velocities of psTatA‐FRET‐digestion versus the corresponding substrate concentration.