Enzymatic activity assays

The enzymatic activity was measured by following the absorbance change induced when a para-nitroanilide (pNA) labeled substrate is enzymatically cleaved using aminoacyl-pNA compounds H-Leu-pNA and H-Leu-Val-Leu-Ala-pNA (Bachem, Bubendorf, Switzerland) as substrates. Measurements were performed on a BioTek Synergy H4 plate reader (Fisher Scientific) measuring the absorbance at 410 nm in a 384-well plate at 50 ^∘C. In all cases, the wells were filled with 50 μL of substrate solution at concentrations varying in the range from 0.1 to 6.4 mM for H-Leu-pNA and 1 mM for H-Leu-Val-Leu-Ala-pNA in buffer (20 mM Tris, 100 mM NaCl, pH 7.5); plates were briefly centrifuged to ensure that the solution is in the bottom of the wells. The plate loaded with the substrate solutions was pre-equilibrated for 20 min at 50 ^∘C. Then, 10 μL of the protein solution (in the same buffer as the substrate) was added on each well in order to reach a final protein solution concentration on each well of 5 ng/μL. All solutions contained 2.8% (vol/vol) dimethylsulfoxide (DMSO; Sigma-Aldrich), which increases the solubility of the substrates. In order to minimize changes in the substrate solution (e.g., temperature) upon the protein addition, the plate was kept above the plate-reader thermostat and an electronic multichannel pipette was employed to load the protein solution into the wells and gently mix the solution. We estimated the pNA concentration from the solution absorptivity (molar absorption coefficient for the pNA at 410 nm of 8800 M⁻¹ cm⁻¹). The path length (0.375 cm) was estimated considering the shape and dimensions of the plate wells and the final volume of the solution. Before analysis, curves from blank sample (no protein) were substracted. The time-dependent absorbance values were analyzed with in-house written python scripts, by fitting the initial rate with a linear equation. Duplicate measurements (time traces of pNA absorbance) were performed. The error estimate of these initial slopes was obtained from the python function lmfit (least-squares fit routine). The difference of the duplicate measurements was small (ca. 3% or less), of the same order as the error estimate. These initial-regime slopes as a function of the substrate concentration were fitted to obtain Michaelis-Menten parameters K_M and k_cat, reported in Figs. 1d and ​and4b,c4b,c and Supplementary Table 1. In this fit, all data points (including duplicates) were used in a joint fit. To determine the error estimates of the K_M and k_cat parameters, a Monte Carlo approach was chosen, following the principles described e.g., in ref. ⁸⁰. In brief, 1000 noisy data sets (initial slope vs. substrate concentration) were created, assuming a normal distribution around the experimentally obtained slopes with σ corresponding to the error estimate of the slope (see above). The reported error bars in Fig. 4b and Supplementary Table ¹ are the standard deviations over these 1000 Monte Carlo fits.

The measurements shown in Supplementary Figures ¹⁷ and ¹⁸ were done with essentially the same approach and minute changes: measurements on the same instrument as above were done in 96-well plates, equally at 50 ^∘C. In all cases, the wells were filled with 80 μL of H-Leu-pNA substrate solution at 6.4 mM in either 20 mM Tris, 100 mM NaCl, pH 7.5, or in 100 mM CAPSO buffer at pH 9.3 or in 100 mM MES buffer at pH 5.3. To investigate the role of free histidine, 200 mM histidine solution was prepared in pH 7.5 buffer and the volume added to the reaction well was adjusted to have final concentration of 1, 5 or 20 mM histidine in the reaction solution.