Enzyme activity of DNA gyrase in the presence of 3. (a) In vivo DNA supercoiling activity when treated with 3 for the indicated times. Treatment with 3 reduced
the
amount of negative supercoiling compared to solvent (D = DMSO) and
TolC inhibitor (P = PAβN) controls. (b) Topo IV activity assay
using kDNA substrates at different concentrations of 3 and 1. +Ctrl indicates the reaction without any inhibitors.
(A) Solvent control (DMSO), (B) 0.078 μM, (C) 0.313 μM,
(D) 1.25 μM, (E) 5 μM, (F) 20 μM, (G) 80 μM,
(H) 160 μM, and (I) 320 μM 3. (J) Solvent
control (50:50 [v/v] of 0.1 M HCl/MeOH), (K) 0.019 μM, (L) 0.078
μM, (M) 0.313 μM, (N) 1.25 μM, (O) 5 μM, (P)
20 μM, (Q) 80 μM, (R) 160 μM 1. (c)
In vitro DNA-dependent ATP hydrolysis in the presence of different
concentrations of 3. Km, Ki, and Vmax were
calculated from the Michaelis–Menten plot shown. ATP assays
were performed in triplicate, and error bars indicate standard deviation
from the mean. Km varies with increasing
concentration of 3; however, Vmax remains unchanged, which is indicative of competitive inhibition.