Fluorometric and Colorimetric Enzyme Activity Assays

4MU substrate assays were performed as previously described (25 (link)). Substrates were used at a final concentration of 10 μM. Enzymes were used at a 150 nM final concentration for the experiments represented in Fig. 1A. Enzymes were used at a final concentration of 50 nM for the experiments represented in Fig. S1B in the supplemental material. Fluorescence (λ_ex = 365 nm and λ_em = 455 nm) was measured every minute on a Cytation 3 imaging reader (BioTek, Winooski, VT, USA) for 60 min.
4-Nitrophenol acetate (4NPA) and 4-nitrophenol thiol-acetate (4-S-NPA) assays were performed as previously described for 4-nitrophenol octanoate (4NPO) (12 (link)). Enzymes were used at a final concentration of 150 nM. Absorbance was monitored on a Cytation 3 imaging reader (BioTek, Winooski, VT, USA).
QStE assays were performed as previously described. In short, a 10 μM final concentration of QstE substrate was incubated with 150 nM enzyme in reaction buffer (20 mM HEPES [pH 7.4], 150 mM NaCl, 10 mM CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate}) at 37°C. Fluorescence (λ_ex = 410 nm and λ_em = 450 nm) was monitored every minute for 1 h on a Cytation 3 imaging reader (BioTek, Winooski, VT, USA).