| Assay Method Information | |
| | FabZ Inhibition Assay |
| Description: | The enzymatic activities of FtFabZ and YpFabZ were determined via the reportedspectrophotometric method using the substrate analogue crotonoyl-CoA.7 Initial reaction velocities were determined by monitoring the decreasing absorbance at 280 nm, resulting from the conversion of crotonoyl-CoA to beta-hydroxybutyryl-CoA. The conversion of hydroxybutyryl-CoA to crotonoyl-CoA was not used in our assay. An extinction coefficient of 3600 s-1 cm-1, corresponding to the hydration of the enoyl double bond, was used to convert absorbance readings to substrate concentrations.20 Reactions were performed using a Beckman-Coulter DU730 UV/vis spectrophotometer. Reaction volumes of 100 uL were prepared in a buffer solution consisting of 2.0 ug of FtFabZ or 0.5 ug of YpFabZ, 20 mM Tris, pH 7.0. Allreactions were performed at 25 C. Inhibition reactions for IC50 calculations wereperformed under similar conditions with a fixed concentration of 100 uM crotonoyl-CoA. |
| Affinity data for this assay | |
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