*2.1.4 Spectrophotometric assay of OPH variant activity*

For cell-free synthesized OPH variants, their activities and substrate binding affinities were measured directly in the cell-free reaction mixture using a BioTek plate reader (Winooski, VT) and that reported light absorbance of the paraoxon leaving group at 405 nm. The assay conditions for cell-free expression were carried out by adding various amounts of paraoxon (final concentration: 0.1, 0.2, 0.5, 1.0, 2.0 and 3.0 mM) to 193.7 μL of buffer containing 100 mM CHES and 75 μM of Zn(OAc)2, at pH = 9.0. The reactions were started by adding 2 μL of cell-free generated WT or OPH variants.

The kinetic assays of OPH UAA variants were performed also by measuring the absorbance of the paraoxon leaving group at 405 nm, as mentioned above. The changes of absorbance at 405 nm over time were used to calculate initial catalytic velocities as a function of the various substrate concentrations. Then, the initial velocity data, along with corresponding paraoxon concentrations, were plotted and analyzed by the Michaelis-Menten equation to obtain *V*max and *K*m using GraphPad (GraphPad Software, Inc., La Jolla, CA, USA). The *k*cat was calculated by the ratio of *V*max and mutant concentration. Each kinetic measurement condition was performed in triplicate and standard deviation was calculated using GraphPad.
