Table 2

Steady-state kinetics of M. smegmatis DprE1 enzymes.

HRP-coupled assay*DCPIP assay*
kcat (min−1)Km (mM)kcat/Km (min−1 mM−1)kcat (min−1)Km (mM)kcat/Km (min−1 mM−1)
Wild type3.1 ± 0.40.19 ± 0.0216.3 ± 2.712.7 ± 0.60.11 ± 0.01115.5 ± 10.9
C394Gn.d.§n.d.n.d.2.9 ± 0.20.36 ± 0.038.0 ± 0.9
Q343An.d.n.d.n.d.3.2 ± 0.10.29 ± 0.0211.0 ± 1.3
K425ANot activeNot active

*All assays were performed at 25°C (at 37°C for progress curves analysis; Fig. 4C) with FPR as substrate, in 20 mM glycylglycine (pH 8.5) because this pH value yielded the best kinetic parameters for enzyme activity. No effect of ionic strength up to 250 mM NaCl was observed.

†With the HRP-coupled assay, the Michaelis-Menten curve showed a sigmoidal shape with a Hill coefficient of 2.0, which might be related to a low reactivity of DprE1 with molecular oxygen (see the text). The substrate concentration at half of the maximum velocity is reported as Km (rather than in the more rigorous form s0.5).

‡The kinetic constants determined for the DCPIP as substrate of the reduced enzyme are Km = 4.2 ± 0.3 μM and kcat = 11.1 ± 0.1 min−1.

§n.d., not determined.