Z ggl amc

Peptidase was assayed using the chromogenic peptide, carbobenzoxy-Gly-Gly-Leu-7-amido-4-methyl coumarin (Z-GGL-AMC; Bachem) as a substrate [30] (link). Wild-type EcU, the P315T, F441Y, P315T/F441Y mutants and two synthetic octapeptides were used for EcV activation. The activity assay was conducted at 37°C in a buffer consisting of 20 mM Tris-HCl pH 7.7, 300 mM NaCl, 1 mM EDTA, 6.25 mM MgCl₂, and 7.5% (v/v) dimethylformamide. When EcU or its mutant enzymes and not a peptide were added as an activator for EcV, ATP was added in the reaction mixture. The release of 7-amido-4-methyl-coumarin (AMC), which is dependent on peptidase activity, was monitored as reported previously [24] (link). For the caseinolytic activity assay, resorufin-labeled casein purchased from Roche Diagnostics, GmbH was used as the substrate [31] . The procedure for activity measurements followed the instructions provided by the manufacturer. For protein substrate degradation, MPB-SulA was used as described previously [7] (link), [24] (link), [32] (link), [33] (link) with some modifications. The ATPase activity of EcU or its mutants was also assayed as described previously [25] , [34] (link). Proteins were quantified by the method of Bradford using bovine serum albumin as a standard [35] (link).

All peptides (Table 1 and Table S1) were assembled on a 2-chloro-trityl resin following either manual or microwave-assisted solid phase synthetic protocols with Fmoc as the Nα-protecting group and HBTU or HATU/DIEA as coupling agents. Their synthesis as well that of the substrate JMV4482 (Z-EVNL-AMC) routinely used for activity tests are described in detail in the Supplementary Materials. Suc-LLVY-AMC and Z-GGL-AMC were purchased from Bachem. pET-Duet-1 plasmid with inducible E. coli C-terminally 6His-tagged HslV were kindly given by Dr Chin Ha Chung from the Department of Biological Sciences of Seoul National University.

Proteolytic activities were measured in a kinetic continuous assay using the following peptidyl fluorogenic, 7-amino-4-methylcoumarin (AMC) substrates (Bachem) at a 50 µM final concentration: Z-F-R-AMC (Z, Benzyloxycarbonyl), Bz-F-V-R-AMC (Bz, Benzoyl), Z-G-P-R-AMC, P-F-R-AMC, Boc-L-R-R-AMC (Boc, t-Butyloxycarbonyl), Boc-Q-A-R-AMC, Boc-V-L-K-AMC Suc-A-A-F-AMC (Suc, Succinyl), Suc-A-A-P-F-AMC, Suc-L-Y-AMC, MeOSuc-A-A-P-V-AMC (MeOSuc, 3-Methoxysuccinyl), Z-G-G-L-AMC and Z-V-K-M-AMC. Assays were performed at 37°C in 96-well black microplates in a total volume of 100 µl. Parasite extracts (1–3 µg) or E/S products (0.05–1 µg) were pre-incubated for 10 min in 150 mM Tris-HCl, pH 8.0, containing 10 µM E64, 1 mM EDTA in the presence or absence of 0.5 mM of the serine protease inhibitors, Pefabloc SC and PMSF. E64 was included routinely in extract preparations in order to inhibit Clan CA cysteine protease activity that is present in the life-stages examined [30] (link), [39] (link), [40] (link). Hydrolysis of substrate was measured continuously using an Infinite M1000 microplate reader (Tecan) at excitation and emission wavelengths of 360 and 465 nm, respectively. All measurements were performed in triplicate and results normalized to protein concentration.