| Assay Method Information | |
| | Fluorescence-Based Competition Assay |
| Description: | A fluorescence-based competition assay was used to determine the kinetic parameters for binding of the inhibitors to paLpxC. This assay is based on the paLpxC ligand PT855, which fluoresces at 420 nm when excited at 325 nm (FIG. 10 ), and whose fluorescence is quenched upon binding to paLpxC (Georgi, 2018). Inhibitors (10 μM for enantiomerically pure compounds or 20 μM for racemic mixtures) were incubated with paLpxC (10 μM) for 18 h at 37° C. to ensure complete formation of the final enzyme-inhibitor complex, and then diluted 200-fold into a 200 nM solution of the fluorescent probe (PT855) at 37° C. The rate of dissociation of the enzyme-inhibitor (EI) complex was determined by monitoring the decrease in fluorescence as a function of time as PT855 displaced the inhibitor from the enzyme. |
| Affinity data for this assay | |
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